53 human secreted proteins

ABSTRACT

The present invention relates to novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating diseases, disorders, and/or conditions related to these novel human secreted proteins.

This application claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 60/244,591, filed Nov. 1, 2000; thisapplication is also a continuation-in-part of, and claims priority under35 U.S.C. §120 to U.S. patent application Ser. No. 09/288,143, filedApr. 8, 1999 now U.S. Pat. No. 6,433,139, which is acontinuation-in-part, and claims priority under 35 U.S.C. §120 toInternational Patent Application No: PCT/US98/21142, filed Oct. 8, 1998(published in English), which claims benefit under 35 U.S.C. §119(e) ofU.S. Provisional Application Nos. 60/061,463, 60/061,529, 60/071,498,60/061,527, 60/061,536, and 60/061,532, filed on Oct. 9, 1997. Each ofthe above referenced applications is hereby incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel proteins. More specifically,isolated nucleic acid molecules are provided encoding novelpolypeptides. Novel polypeptides and antibodies that bind to thesepolypeptides are provided. Also provided are vectors, host cells, andrecombinant and synthetic methods for producing human polynucleotidesand/or polypeptides, and antibodies. The invention further relates todiagnostic and therapeutic methods useful for diagnosing, treating,preventing and/or prognosing disorders related to these novelpolypeptides. The invention further relates to screening methods foridentifying agonists and antagonists of polynucleotides and polypeptidesof the invention. The present invention further relates to methodsand/or compositions for inhibiting or enhancing the production andfunction of the polypeptides of the present invention.

BACKGROUND OF THE INVENTION

Unlike bacterium, which exist as a single compartment surrounded by amembrane, human cells and other eukaryotes are subdivided by membranesinto many functionally distinct compartments. Each membrane-boundedcompartment, or organelle, contains different proteins essential for thefunction of the organelle. The cell uses “sorting signals,” which areamino acid motifs located within the protein, to target proteins toparticular cellular organelles.

One type of sorting signal, called a signal sequence, a signal peptide,or a leader sequence, directs a class of proteins to an organelle calledthe endoplasmic reticulum (ER). The ER separates the membrane-boundedproteins from all other types of proteins. Once localized to the ER,both groups of proteins can be further directed to another organellecalled the Golgi apparatus. Here, the Golgi distributes the proteins tovesicles, including secretory vesicles, the cell membrane, lysosomes,and the other organelles.

Proteins targeted to the ER by a signal sequence can be released intothe extracellular space as a secreted protein. For example, vesiclescontaining secreted proteins can fuse with the cell membrane and releasetheir contents into the extracellular space—a process called exocytosis.Exocytosis can occur constitutively or after receipt of a triggeringsignal. In the latter case, the proteins are stored in secretoryvesicles (or secretory granules) until exocytosis is triggered.Similarly, proteins residing on the cell membrane can also be secretedinto the extracellular space by proteolytic cleavage of a “linker”holding the protein to the membrane.

Thus there exists a clear need for identifying and using novel secretedpolynucleotides and polypeptides. Identification and sequencing of humangenes is a major goal of modern scientific research. For example, byidentifying genes and determining their sequences, scientists have beenable to make large quantities of valuable human “gene products.” Theseinclude human insulin, interferon, Factor VIII, tumor necrosis factor,human growth hormone, tissue plasminogen activator, and numerous othercompounds. Additionally, knowledge of gene sequences can provide the keyto treatment or cure of genetic diseases (such as muscular dystrophy andcystic fibrosis).

SUMMARY OF THE INVENTION

The present invention relates to novel secreted proteins. Morespecifically, isolated nucleic acid molecules are provided encodingnovel secreted polypeptides. Novel polypeptides and antibodies that bindto these polypeptides are provided. Also provided are vectors, hostcells, and recombinant and synthetic methods for producing humanpolynucleotides and/or polypeptides, and antibodies. The inventionfurther relates to diagnostic and therapeutic methods useful fordiagnosing, treating, preventing and/or prognosing disorders related tothese novel polypeptides. The invention further relates to screeningmethods for identifying agonists and antagonists of polynucleotides andpolypeptides of the invention. The present invention further relates tomethods and/or compositions for inhibiting or enhancing the productionand function of the polypeptides of the present invention.

DETAILED DESCRIPTION

Polynucleotides and Polypeptides

Description of Table 1A

Table 1A summarizes information concerning certain polynucleotides andpolypeptides of the invention. The first column provides the gene numberin the application for each clone identifier. The second column providesa unique clone identifier, “Clone ID”, for a cDNA clone related to eachcontig sequence disclosed in Table 1A. Third column, the cDNA Clonesidentified in the second column were deposited as indicated in the thirdcolumn (i.e. by ATCC Deposit Number and deposit date). Some of thedeposits contain multiple different clones corresponding to the samegene. In the fourth column, “Vector” refers to the type of vectorcontained in the corresponding cDNA Clone identified in the secondcolumn. In the fifth column, the nucleotide sequence identified as “NTSEQ ID NO:X” was assembled from partially homologous (“overlapping”)sequences obtained from the corresponding cDNA clone identified in thesecond column and, in some cases, from additional related cDNA clones.The overlapping sequences were assembled into a single contiguoussequence of high redundancy (usually three to five overlapping sequencesat each nucleotide position), resulting in a final sequence identifiedas SEQ ID NO:X. In the sixth column, “Total NT Seq.” refers to the totalnumber of nucleotides in the contig sequence identified as SEQ ID NO:X.”The deposited clone may contain all or most of these sequences,reflected by the nucleotide position indicated as “5′ NT of Clone Seq.”(seventh column) and the “3′ NT of Clone Seq.” (eighth column) of SEQ IDNO:X. In the ninth column, the nucleotide position of SEQ ID NO:X of theputative start codon (methionine) is identified as “5′ NT of StartCodon.” Similarly, in column ten, the nucleotide position of SEQ ID NO:Xof the predicted signal sequence is identified as “5′ NT of First AA ofSignal Pep.” In the eleventh column, the translated amino acid sequence,beginning with the methionine, is identified as “AA SEQ ID NO:Y,”although other reading frames can also be routinely translated usingknown molecular biology techniques. The polypeptides produced by thesealternative open reading frames are specifically contemplated by thepresent invention.

In the twelfth and thirteenth columns of Table 1A, the first and lastamino acid position of SEQ ID NO:Y of the predicted signal peptide isidentified as “First AA of Sig Pep” and “Last AA of Sig Pep.” In thefourteenth column, the predicted first amino acid position of SEQ IDNO:Y of the secreted portion is identified as “Predicted First AA ofSecreted Portion”. The amino acid position of SEQ ID NO:Y of the lastamino acid encoded by the open reading frame is identified in thefifteenth column as “Last AA of ORF”.

SEQ ID NO:X (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:Y (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further below. For instance,SEQ ID NO:X is useful for designing nucleic acid hybridization probesthat will detect nucleic acid sequences contained in SEQ ID NO:X or thecDNA contained in the deposited clone. These probes will also hybridizeto nucleic acid molecules in biological samples, thereby enabling avariety of forensic and diagnostic methods of the invention. Similarly,polypeptides identified from SEQ ID NO:Y may be used, for example, togenerate antibodies which bind specifically to proteins containing thepolypeptides and the secreted proteins encoded by the cDNA clonesidentified in Table 1A and/or elsewhere herein

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:X, and the predicted translated amino acid sequence identified as SEQID NO:Y, but also a sample of plasmid DNA containing a human cDNA of theinvention deposited with the ATCC, as set forth in Table 1A. Thenucleotide sequence of each deposited plasmid can readily be determinedby sequencing the deposited plasmid in accordance with known methods

The predicted amino acid sequence can then be verified from suchdeposits. Moreover, the amino acid sequence of the protein encoded by aparticular plasmid can also be directly determined by peptide sequencingor by expressing the protein in a suitable host cell containing thedeposited human cDNA, collecting the protein, and determining itssequence.

Also provided in Table 1A is the name of the vector which contains thecDNA plasmid. Each vector is routinely used in the art. The followingadditional information is provided for convenience.

Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR(U.S. Pat. Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Pat. Nos.5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al.,Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J.M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. etal., Strategies 5:58-61 (1992)) are commercially available fromStratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla,Calif., 92037. pBS contains an ampicillin resistance gene and pBKcontains a neomycin resistance gene. Phagemid pBS may be excised fromthe Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excisedfrom the Zap Express vector. Both phagemids may be transformed into E.coli strain XL-1 Blue, also available from Stratagene.

Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, wereobtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, Md.20897. All Sport vectors contain an ampicillin resistance gene and maybe transformed into E. coli strain DH10B, also available from LifeTechnologies. See, for instance, Gruber, C. E., et al., Focus 15:59(1993). Vector lafmid BA (Bento Soares, Columbia University, New York,N.Y.) contains an ampicillin resistance gene and can be transformed intoE. coli strain XL-1 Blue. Vector pCR®2.1, which is available fromInvitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains anampicillin resistance gene and may be transformed into E. coli strainDH10B, available from Life Technologies. See, for instance, Clark, J.M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al.,Bio/Technology 9: (1991).

The present invention also relates to the genes corresponding to SEQ IDNO:X, SEQ ID NO:Y, and/or a deposited cDNA (Clone ID). The correspondinggene can be isolated in accordance with known methods using the sequenceinformation disclosed herein. Such methods include, but are not limitedto, preparing probes or primers from the disclosed sequence andidentifying or amplifying the corresponding gene from appropriatesources of genomic material.

Also provided in the present invention are allelic variants, orthologs,and/or species homologs. Procedures known in the art can be used toobtain full-length genes, allelic variants, splice variants, full-lengthcoding portions, orthologs, and/or species homologs of genescorresponding to SEQ ID NO:X and SEQ ID NO:Y using information from thesequences disclosed herein or the clones deposited with the ATCC. Forexample, allelic variants and/or species homologs may be isolated andidentified by making suitable probes or primers from the sequencesprovided herein and screening a suitable nucleic acid source for allelicvariants and/or the desired homologue.

The present invention provides a polynucleotide comprising, oralternatively consisting of, the nucleic acid sequence of SEQ ID NO:Xand/or a cDNA contained in ATCC Deposit No. Z. The present inventionalso provides a polypeptide comprising, or alternatively, consisting of,the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ IDNO:X, and/or a polypeptide encoded by a cDNA contained in ATCC depositNo. Z. Polynucleotides encoding a polypeptide comprising, oralternatively consisting of the polypeptide sequence of SEQ ID NO:Y, apolypeptide encoded by SEQ ID NO:X and/or a polypeptide encoded by thecDNA contained in ATCC Deposit No. Z, are also encompassed by theinvention. The present invention further encompasses a polynucleotidecomprising, or alternatively consisting of the complement of the nucleicacid sequence of SEQ ID NO:X, and/or the complement of the coding strandof the cDNA contained in ATCC Deposit No. Z.

Description of Table 1B

Table 1B summarizes some of the polynucleotides encompassed by theinvention (including cDNA clones related to the sequences (Clone ID),contig sequences (contig identifier (Contig ID:) and contig nucleotidesequence identifier (SEQ ID NO:X)) and further summarizes certaincharacteristics of these polynucleotides and the polypeptides encodedthereby. The first column provides the gene number in the applicationfor each clone identifier. The second column provides a unique cloneidentifier, “Clone ID”, for a cDNA clone related to each contig sequencedisclosed in Table 1A and/or 1B. The third column provides a uniquecontig identifier, “Contig ID:” for each of the contig sequencesdisclosed in Table 1B. The fourth column provides the sequenceidentifier, “SEQ ID NO:X”, for each of the contig sequences disclosed inTable 1A and/or 1B. The fifth column, “ORF (From-To)”, provides thelocation (i.e., nucleotide position numbers) within the polynucleotidesequence of SEQ ID NO:X that delineate the preferred open reading frame(ORF) that encodes the amino acid sequence shown in the sequence listingand referenced in Table 1B as SEQ ID NO:Y (column 6). Column 7 listsresidues comprising predicted epitopes contained in the polypeptidesencoded by each of the preferred ORFs (SEQ ID NO:Y). Identification ofpotential immunogenic regions was performed according to the method ofJameson and Wolf (CABIOS, 4; 181-186 (1988)); specifically, the GeneticsComputer Group (GCG) implementation of this algorithm, embodied in theprogram PEPTIDESTRUCTURE (Wisconsin Package v10.0, Genetics ComputerGroup (GCG), Madison, Wis.). This method returns a measure of theprobability that a given residue is found on the surface of the protein.Regions where the antigenic index score is greater than 0.9 over atleast 6 amino acids are indicated in Table 1B as “Predicted Epitopes”.In particular embodiments, polypeptides of the invention comprise, oralternatively consist of, one, two, three, four, five or more of thepredicted epitopes described in Table 1B. It will be appreciated thatdepending on the analytical criteria used to predict antigenicdeterminants, the exact address of the determinant may vary slightly.Column 8, “Tissue Distribution” shows the expression profile of tissue,cells, and/or cell line libraries which express the polynucleotides ofthe invention. The first number in column 8 (preceding the colon),represents the tissue/cell source identifier code corresponding to thekey provided in Table 4. Expression of these polynucleotides was notobserved in the other tissues and/or cell libraries tested. For thoseidentifier codes in which the first two letters are not “AR”, the secondnumber in column 8 (following the colon), represents the number of timesa sequence corresponding to the reference polynucleotide sequence (e.g.,SEQ ID NO:X) was identified in the tissue/cell source. Those tissue/cellsource identifier codes in which the first two letters are “AR”designate information generated using DNA array technology. Utilizingthis technology, cDNAs were amplified by PCR and then transferred, induplicate, onto the array. Gene expression was assayed throughhybridization of first strand cDNA probes to the DNA array. cDNA probeswere generated from total RNA extracted from a variety of differenttissues and cell lines. Probe synthesis was performed in the presence of³³P dCTP, using oligo(dT) to prime reverse transcription. Afterhybridization, high stringency washing conditions were employed toremove non-specific hybrids from the array. The remaining signal,emanating from each gene target, was measured using a Phosphorimager.Gene expression was reported as Phosphor Stimulating Luminescence (PSL)which reflects the level of phosphor signal generated from the probehybridized to each of the gene targets represented on the array. A localbackground signal subtraction was performed before the total signalgenerated from each array was used to normalize gene expression betweenthe different hybridizations. The value presented after “[array code]:”represents the mean of the duplicate values, following backgroundsubtraction and probe normalization. One of skill in the art couldroutinely use this information to identify normal and/or diseasedtissue(s) which show a predominant expression pattern of thecorresponding polynucleotide of the invention or to identifypolynucleotides which show predominant and/or specific tissue and/orcell expression. Column 9 provides the chromosomal location ofpolynucleotides corresponding to SEQ ID NO:X. Chromosomal location wasdetermined by finding exact matches to EST and cDNA sequences containedin the NCBI (National Center for Biotechnology Information) UniGenedatabase. Given a presumptive chromosomal location, disease locusassociation was determined by comparison with the Morbid Map, derivedfrom Online Mendelian Inheritance in Man (Online Mendelian Inheritancein Man, OMIM™. McKusick-Nathans Institute for Genetic Medicine, JohnsHopkins University (Baltimore, Md.) and National Center forBiotechnology Information, National Library of Medicine (Bethesda, Md.)2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/). If theputative chromosomal location of the Query overlaps with the chromosomallocation of a Morbid Map entry, an OMIM identification number isdisclosed in column 10 labeled “OMIM Disease Reference(s)”. A key to theOMIM reference identification numbers is provided in Table 5.Description of Table 1CTable 1C summarizes additional polynucleotides encompassed by theinvention (including cDNA clones related to the sequences (Clone ID),contig sequences (contig identifier (Contig ID:) contig nucleotidesequence identifiers (SEQ ID NO:X)), and genomic sequences (SEQ IDNO:B). The first column provides a unique clone identifier, “Clone ID”,for a cDNA clone related to each contig sequence. The second columnprovides the sequence identifier, “SEQ ID NO:X”, for each contigsequence. The third column provides a unique contig identifier, “ContigID:” for each contig sequence. The fourth column, provides a BACidentifier “BAC ID NO:A” for the BAC clone referenced in thecorresponding row of the table. The fifth column provides the nucleotidesequence identifier, “SEQ ID NO:B” for a fragment of the BAC cloneidentified in column four of the corresponding row of the table. Thesixth column, “Exon From-To”, provides the location (i.e., nucleotideposition numbers) within the polynucleotide sequence of SEQ ID NO:Bwhich delineate certain polynucleotides of the invention that are alsoexemplary members of polynucleotide sequences that encode polypeptidesof the invention (e.g., polypeptides containing amino acid sequencesencoded by the polynucleotide sequences delineated in column six, andfragments and variants thereof).Description of Table 1DTable 1D: In preferred embodiments, the present invention encompasses amethod of treating a disease or disorder listed in the “FEATURES OFPROTEIN” sections (below) and also as listed in the “PreferredIndications” column of Table 1D (below); comprising administering to apatient in which such treatment, prevention, or amelioration is desireda protein, nucleic acid, or antibody of the invention (or fragment orvariant thereof) represented by Table 1A and Table 1D (in the same rowas the disease or disorder to be treated is listed in the “PreferredIndications” column of Table 1D) in an amount effective to treat,prevent, or ameliorate the disease or disorder.

As indicated in Table 1D, the polynucleotides, polypeptides, agonists,or antagonists of the present invention (including antibodies) can beused in assays to test for one or more biological activities. If thesepolynucleotides and polypeptides do exhibit activity in a particularassay, it is likely that these molecules may be involved in the diseasesassociated with the biological activity. Thus, the polynucleotides orpolypeptides, or agonists or antagonists thereof (including antibodies)could be used to treat the associated disease.

The present invention encompasses methods of preventing, treating,diagnosing, or ameliorating a disease or disorder. In preferredembodiments, the present invention encompasses a method of treating adisease or disorder listed in the “Preferred Indications” column ofTable 1D; comprising administering to a patient in which such treatment,prevention, or amelioration is desired a protein, nucleic acid, orantibody of the invention (or fragment or variant thereof) in an amounteffective to treat, prevent, diagnose, or ameliorate the disease ordisorder. The first and second columns of Table 1D show the “Gene No.”and “Clone ID”, respectively, indicating certain nucleic acids andproteins (or antibodies against the same) of the invention (includingpolynucleotide, polypeptide, and antibody fragments or variants thereof)that may be used in preventing, treating, diagnosing, or amelioratingthe disease(s) or disorder(s) indicated in the corresponding row inColumn 3 of Table 1D.

In another embodiment, the present invention also encompasses methods ofpreventing, treating, diagnosing, or ameliorating a disease or disorderlisted in the “Preferred Indications” column of Table 1D; comprisingadministering to a patient combinations of the proteins, nucleic acids,or antibodies of the invention (or fragments or variants thereof),sharing similar indications as shown in the corresponding rows in Column3 of Table 1D.

The “Preferred Indication” column describes diseases, disorders, and/orconditions that may be treated, prevented, diagnosed, or ameliorated bya protein, nucleic acid, or antibody of the invention (or fragment orvariant thereof).

The recitation of “Cancer” in the “Preferred Indication” columnindicates that the corresponding nucleic acid and protein, or antibodyagainst the same, of the invention (or fragment or variant thereof) maybe used for example, to diagnose, treat, prevent, and/or amelioratediseases and/or disorders relating to neoplastic diseases (e.g.,leukemias, cancers, and/or as described below under “HyperproliferativeDisorders”).

In specific embodiments, a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) having a “Cancer” recitationin the “Preferred Indication” column of Table 1D may be used forexample, to diagnose, treat, prevent, and/or ameliorate a neoplasmlocated in a tissue selected from the group consisting of: colon,abdomen, bone, breast, digestive system, liver, pancreas, prostate,peritoneum, lung, blood (e.g., leukemia), endocrine glands (adrenal,parathyroid, pituitary, testicles, ovary, thymus, thyroid), uterus, eye,head and neck, nervous (central and peripheral), lymphatic system,pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

In specific embodiments, a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) having a “Cancer” recitationin the “Preferred Indication” column of Table 1D, may be used forexample, to diagnose, treat, prevent, and/or ameliorate a pre-neoplasticcondition, selected from the group consisting of: hyperplasia (e.g.,endometrial hyperplasia and/or as described in the section entitled“Hyperproliferative Disorders”), metaplasia (e.g., connective tissuemetaplasia, atypical metaplasia, and/or as described in the sectionentitled “Hyperproliferative Disorders”), and/or dysplasia (e.g.,cervical dysplasia, and bronchopulmonary dysplasia).

In another specific embodiment, a protein, nucleic acid, or antibody ofthe invention (or fragment or variant thereof) having a “Cancer”recitation in the “Preferred Indication” column of Table 1D, may be usedfor example, to diagnose, treat, prevent, and/or ameliorate a benigndysproliferative disorder selected from the group consisting of: benigntumors, fibrocystic conditions, tissue hypertrophy, and/or as describedin the section entitled “Hyperproliferative Disorders”.

The recitation of “Immune/Hematopoietic” in the “Preferred Indication”column indicates that the corresponding nucleic acid and protein, orantibody against the same, of the invention (or fragment or variantthereof), may be used for example, to diagnose, treat, prevent, and/orameliorate diseases and/or disorders relating to neoplastic diseases(e.g., as described below under “Hyperproliferative Disorders”), blooddisorders (e.g., as described below under “Immune Activity”“Cardiovascular Disorders” and/or “Blood-Related Disorders”), andinfections (e.g., as described below under “Infectious Disease”).

In specific embodiments, a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) having the“Immune/Hematopoietic” recitation in the “Preferred Indication” columnof Table 1D, may be used for example, to diagnose, treat, prevent,and/or ameliorate a disease or disorder selected from the groupconsisting of: anemia, pancytopenia, leukopenia, thrombocytopenia,leukemias, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocyticanemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma,arthritis, asthma, AIDS, autoimmnune disease, rheumatoid arthritis,granulomatous disease, immune deficiency, inflammatory bowel disease,sepsis, neutropenia, neutrophilia, psoriasis, immune reactions totransplanted organs and tissues, systemic lupus erythematosis,hemophilia, hypercoagulation, diabetes mellitus, endocarditis,meningitis, Lyme Disease, and allergies.

The recitation of “Reproductive” in the “Preferred Indication” columnindicates that the corresponding nucleic acid and protein, or antibodyagainst the same, of the invention (or fragment or variant thereof), maybe used for example, to diagnose, treat, prevent, and/or amelioratediseases and/or disorders relating to neoplastic diseases (e.g., asdescribed below under “Hyperproliferative Disorders”), and disorders ofthe reproductive system (e.g., as described below under “ReproductiveSystem Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) having a “Reproductive”recitation in the “Preferred Indication” column of Table 1D, may be usedfor example, to diagnose, treat, prevent, and/or ameliorate a disease ordisorder selected from the group consisting of: cryptorchism,prostatitis, inguinal hernia, varicocele, leydig cell tumors, verrucouscarcinoma, prostatitis, malacoplakia, Peyronie's disease, penilecarcinoma, squamous cell hyperplasia, dysmenorrhea, ovarianadenocarcinoma, Turner's syndrome, mucopurulent cervicitis,Sertoli-leydig tumors, ovarian cancer, uterine cancer, pelvicinflammatory disease, testicular cancer, prostate cancer, Klinefelter'ssyndrome, Young's syndrome, premature ejaculation, diabetes mellitus,cystic fibrosis, Kartagener's syndrome, testicular atrophy, testicularfeminization, anorchia, ectopic testis, epididymitis, orchitis,gonorrhea, syphilis, testicular torsion, vasitis nodosa, germ celltumors, stromal tumors, dysmenorrhea, retroverted uterus, endometriosis,fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing'ssyndrome, hydatidiform moles, Asherman's syndrome, premature menopause,precocious puberty, uterine polyps, dysfunctional uterine bleeding,cervicitis, chronic cervicitis, mucopurulent cervicitis, cervicaldysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervicalincompetence, cervical neoplasms, pseudohermaphroditism, andpremenstrual syndrome.

The recitation of “Musculoskeletal” in the “Preferred Indication” columnindicates that the corresponding nucleic acid and protein, or antibodyagainst the same, of the invention (or fragment or variant thereof), maybe used for example, to diagnose, treat, prevent, and/or amelioratediseases and/or disorders relating to neoplastic diseases (e.g., asdescribed below under “Hyperproliferative Disorders”), and disorders ofthe immune system (e.g., as described below under “Immune Activity”).

In specific embodiments, a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) having a “Musculoskeletal”recitation in the “Preferred Indication” column of Table 1D, may be usedfor example, to diagnose, treat, prevent, and/or ameliorate a disease ordisorder selected from the group consisting of: bone cancers (e.g.,osteochondromas, benign chondromas, chondroblastoma, chondromyxoidfibromas, osteoid osteomas, giant cell tumors, multiple myeloma,osteosarcomas), Paget's Disease, rheumatoid arthritis, systemic lupuserythematosus, osteomyelitis, Lyme Disease, gout, bursitis, tendonitis,osteoporosis, osteoarthritis, muscular dystrophy, mitochondrialmyopathy, cachexia, and multiple sclerosis.

The recitation of “Cardiovascular” in the “Preferred Indication” columnindicates that the corresponding nucleic acid and protein, or antibodyagainst the same, of the invention (or fragment or variant thereof), maybe used for example, to diagnose, treat, prevent, and/or amelioratediseases and/or disorders relating to neoplastic diseases (e.g., asdescribed below under “Hyperproliferative Disorders”), and disorders ofthe cardiovascular system (e.g., as described below under“Cardiovascular Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) having a “Cardiovascular”recitation in the “Preferred Indication” column of Table 1D, may be usedfor example, to diagnose, treat, prevent, and/or ameliorate a disease ordisorder selected from the group consisting of: myxomas, fibromas,rhabdomyomas, cardiovascular abnormalities (e.g., congenital heartdefects, cerebral arteriovenous malformations, septal defects), heartdisease (e.g., heart failure, congestive heart disease, arrhythmia,tachycardia, fibrillation, pericardial Disease, endocarditis), cardiacarrest, heart valve disease (e.g., stenosis, regurgitation, prolapse),vascular disease (e.g., hypertension, coronary artery disease, angina,aneurysm, arteriosclerosis, peripheral vascular disease), hyponatremia,hypernatremia, hypokalemia, and hyperkalemia.

The recitation of “Mixed Fetal” in the “Preferred Indication” columnindicates that the corresponding nucleic acid and protein, or antibodyagainst the same, of the invention (or fragment or variant thereof), maybe used for example, to diagnose, treat, prevent, and/or amelioratediseases and/or disorders relating to neoplastic diseases (e.g., asdescribed below under “Hyperproliferative Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) having a “Mixed Fetal”recitation in the “Preferred Indication” column of Table 1D, may be usedfor example, to diagnose, treat, prevent, and/or ameliorate a disease ordisorder selected from the group consisting of: spina bifida,hydranencephaly, neurofibromatosis, fetal alcohol syndrome, diabetesmellitus, PKU, Down's syndrome, Patau syndrome, Edwards syndrome, Turnersyndrome, Apert syndrome, Carpenter syndrome, Conradi syndrome, Crouzonsyndrome, cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveldsyndrome, Holt-Oram syndrome, Kartagener syndrome, Meckel-Grubersyndrome, Noonan syndrome, Pallister-Hall syndrome, Rubinstein-Taybisyndrome, Scimitar syndrome, Smith-Lemli-Opitz syndrome,thromocytopenia-absent radius (TAR) syndrome, Treacher Collins syndrome,Williams syndrome, Hirschsprung's disease, Meckel's diverticulum,polycystic kidney disease, Turner's syndrome, and gonadal dysgenesis,Klippel-Feil syndrome, Ostogenesis imperfecta, muscular dystrophy,Tay-Sachs disease, Wilm's tumor, neuroblastoma, and retinoblastoma.

The recitation of “Excretory” in the “Preferred Indication” columnindicates that the corresponding nucleic acid and protein, or antibodyagainst the same, of the invention (or fragment or variant thereof), maybe used for example, to diagnose, treat, prevent, and/or amelioratediseases and/or disorders relating to neoplastic diseases (e.g., asdescribed below under “Hyperproliferative Disorders”) and renaldisorders (e.g., as described below under “Renal Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) having a “Excretory”recitation in the “Preferred Indication” column of Table 1D, may be usedfor example, to diagnose, treat, prevent, and/or ameliorate a disease ordisorder selected from the group consisting of: bladder cancer, prostatecancer, benign prostatic hyperplasia, bladder disorders (e.g., urinaryincontinence, urinary retention, urinary obstruction, urinary tractInfections, interstitial cystitis, prostatitis, neurogenic bladder,hematuria), renal disorders (e.g., hydronephrosis, proteinuria, renalfailure, pyelonephritis, urolithiasis, reflux nephropathy, andunilateral obstructive uropathy).

The recitation of “Neural/Sensory” in the “Preferred Indication” columnindicates that the corresponding nucleic acid and protein, or antibodyagainst the same, of the invention (or fragment or variant thereof), maybe used for example, to diagnose, treat, prevent, and/or amelioratediseases and/or disorders relating to neoplastic diseases (e.g., asdescribed below under “Hyperproliferative Disorders”) and diseases ordisorders of the nervous system (e.g., as described below under “NeuralActivity and Neurological Diseases”).

In specific embodiments, a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) having a “Neural/Sensory”recitation in the “Preferred Indication” column of Table 1D, may be usedfor example, to diagnose, treat, prevent, and/or ameliorate a disease ordisorder selected from the group consisting of: brain cancer (e.g.,brain stem glioma, brain tumors, central nervous system (Primary)lymphoma, central nervous system lymphoma, cerebellar astrocytoma, andcerebral astrocytoma, neurodegenerative disorders (e.g., Alzheimer'sDisease, Creutzfeldt-Jakob Disease, Parkinson's Disease, and IdiopathicPresenile Dementia), encephalomyelitis, cerebral malaria, meningitis,metabolic brain diseases (e.g., phenylketonuria and pyruvate carboxylasedeficiency), cerebellar ataxia, ataxia telangiectasia, and AIDS DementiaComplex, schizophrenia, attention deficit disorder, hyperactiveattention deficit disorder, autism, and obsessive compulsive disorders.

The recitation of “Respiratory” in the “Preferred Indication” columnindicates that the corresponding nucleic acid and protein, or antibodyagainst the same, of the invention (or fragment or variant thereof), maybe used for example, to diagnose, treat, prevent, and/or amelioratediseases and/or disorders relating to neoplastic diseases (e.g., asdescribed below under “Hyperproliferative Disorders”) and diseases ordisorders of the respiratory system (e.g., as described below under“Respiratory Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) having a “Respiratory”recitation in the “Preferred Indication” column of Table 1D, may be usedfor example, to diagnose, treat, prevent, and/or ameliorate a disease ordisorder selected from the group consisting of: cancers of therespiratory system such as larynx cancer, pharynx cancer, tracheacancer, epiglottis cancer, lung cancer, squamous cell carcinomas, smallcell (oat cell) carcinomas, large cell carcinomas, and adenocarcinomas.Allergic reactions, cystic fibrosis, sarcoidosis, histiocytosis X,infiltrative lung diseases (e.g., pulmonary fibrosis and lymphoidinterstitial pneumonia), obstructive airway diseases (e.g., asthma,emphysema, chronic or acute bronchitis), occupational lung diseases(e.g., silicosis and asbestosis), pneumonia, and pleurisy.

The recitation of “Endocrine” in the “Preferred Indication” columnindicates that the corresponding nucleic acid and protein, or antibodyagainst the same, of the invention (or fragment or variant thereof), maybe used for example, to diagnose, treat, prevent, and/or amelioratediseases and/or disorders relating to neoplastic diseases (e.g., asdescribed below under “Hyperproliferative Disorders”) and diseases ordisorders of the respiratory system (e.g., as described below under“Respiratory Disorders”), renal disorders (e.g., as described belowunder “Renal Disorders”), and disorders of the endocrine system (e.g.,as described below under “Endocrine Disorders”.

In specific embodiments, a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) having an “Endocrine”recitation in the “Preferred Indication” column of Table 1D, may be usedfor example, to diagnose, treat, prevent, and/or ameliorate a disease ordisorder selected from the group consisting of: cancers of endocrinetissues and organs (e.g., cancers of the hypothalamus, pituitary gland,thyroid gland, parathyroid glands, pancreas, adrenal glands, ovaries,and testes), diabetes (e.g., diabetes insipidus, type I and type IIdiabetes mellitus), obesity, disorders related to pituitary glands(e.g., hyperpituitarism, hypopituitarism, and pituitary dwarfism),hypothyroidism, hyperthyroidism, goiter, reproductive disorders (e.g.male and female infertility), disorders related to adrenal glands (e.g.,Addison's Disease, corticosteroid deficiency, and Cushing's Syndrome),kidney cancer (e.g., hypernephroma, transitional cell cancer, and Wilm'stumor), diabetic nephropathy, interstitial nephritis, polycystic kidneydisease, glomerulonephritis (e.g., IgM mesangial proliferativeglomerulonephritis and glomerulonephritis caused by autoimmunedisorders; such as Goodpasture's syndrome), and nephrocalcinosis.

The recitation of “Digestive” in the “Preferred Indication” columnindicates that the corresponding nucleic acid and protein, or antibodyagainst the same, of the invention (or fragment or variant thereof), maybe used for example, to diagnose, treat, prevent, and/or amelioratediseases and/or disorders relating to neoplastic diseases (e.g., asdescribed below under “Hyperproliferative Disorders”) and diseases ordisorders of the gastrointestinal system (e.g., as described below under“Gastrointestinal Disorders”.

In specific embodiments, a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) having a “Digestive”recitation in the “Preferred Indication” column of Table 1D, may be usedfor example, to diagnose, treat, prevent, and/or ameliorate a disease ordisorder selected from the group consisting of: ulcerative colitis,appendicitis, Crohn's disease, hepatitis, hepatic encephalopathy, portalhypertension, cholelithiasis, cancer of the digestive system (e.g.,biliary tract cancer, stomach cancer, colon cancer, gastric cancer,pancreatic cancer, cancer of the bile duct, tumors of the colon (e.g.,polyps or cancers), and cirrhosis), pancreatitis, ulcerative disease,pyloric stenosis, gastroenteritis, gastritis, gastric atropy, benigntumors of the duodenum, distension, irritable bowel syndrome,malabsorption, congenital disorders of the small intestine, bacterialand parasitic infection, megacolon, Hirschsprung's disease, aganglionicmegacolon, acquired megacolon, colitis, anorectal disorders (e.g., analfistulas, hemorrhoids), congenital disorders of the liver (e.g.,Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, andalpha1-antitrypsin deficiency), portal hypertension, cholelithiasis, andjaundice.

The recitation of “Connective/Epithelial” in the “Preferred Indication”column indicates that the corresponding nucleic acid and protein, orantibody against the same, of the invention (or fragment or variantthereof), may be used for example, to diagnose, treat, prevent, and/orameliorate diseases and/or disorders relating to neoplastic diseases(e.g., as described below under “Hyperproliferative Disorders”),cellular and genetic abnormalities (e.g., as described below under“Diseases at the Cellular Level”), angiogenesis (e.g., as describedbelow under “Anti-Angiogenesis Activity”), and or to promote or inhibitregeneration (e.g., as described below under “Regeneration”), and woundhealing (e.g., as described below under “Wound Healing and EpithelialCell Proliferation”).

In specific embodiments, a protein, nucleic acid, or antibody of theinvention (or fragment or variant thereof) having a“Connective/Epithelial” recitation in the “Preferred Indication” columnof Table 1D, may be used for example, to diagnose, treat, prevent,and/or ameliorate a disease or disorder selected from the groupconsisting of: connective tissue metaplasia, mixed connective tissuedisease, focal epithelial hyperplasia, epithelial metaplasia,mucoepithelial dysplasia, graft v. host disease, polymyositis, cystichyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer'sdisease, lymphoproliferative disorder, Waldenstron's macroglobulinemia,Crohn's disease, pernicious anemia, idiopathic Addison's disease,glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetesmellitus, cystic fibrosis, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, osteoporosis, osteocarthritis, periodontal disease,wound healing, relapsing polychondritis, vasculitis, polyarteritisnodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis,psoriatic arthritis, discoid lupus erythematosus, systemic lupuserythematosus, scleroderma, CREST syndrome, Sjogren's syndrome,polymyositis, dermatomyositis, mixed connective tissue disease,relapsing polychondritis, vasculitis, Henoch-Schonlein syndrome,erythema nodosum, polyarteritis nodosa, temporal (giant cell) arteritis,Takayasu's arteritis, Wegener's granulomatosis, Reiter's syndrome,Behcet's syndrome, ankylosing spondylitis, cellulitis, keloids, EhlerDanlos syndrome, Marfan syndrome, pseudoxantoma elasticum, osteogeneseimperfecta, chondrodysplasias, epidermolysis bullosa, Alport syndrome,and cutis laxa.

Description of Table 1E

Table 1E provides information related to biological activities andpreferred indications for polynucleotides and polypeptides of theinvention (including antibodies, agonists, and/or antagonists thereof).Table 1E also provides information related to assays which may be usedto test polynucleotides and polypeptides of the invention (includingantibodies, agonists, and/or antagonists thereof) for the correspondingbiological activities. The first column (“Gene No.”) provides the genenumber in the application for each clone identifier. The second column(“Clone ID”) provides the unique clone identifier for each clone aspreviously described and indicated in Tables 1A, 1B, 1C, and 1D. Thethird column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ IDNumber for polypeptide sequences encoded by the corresponding cDNAclones (also as indicated in Tables 1A, 1B, and 2). The fourth column(“Biological Activity”) indicates a biological activity corresponding tothe indicated polypeptides (or polynucleotides encoding saidpolypeptides). The fifth column (“Exemplary Activity Assay”) furtherdescribes the corresponding biological activity and provides informationpertaining to the various types of assays which may be performed totest, demonstrate, or quantify the corresponding biological activity.The sixth column (“Preferred Indications”) describes particularembodiments of the invention and indications (e.g. pathologies,diseases, disorders, abnormalities, etc.) for which polynucleotides andpolypeptides of the invention (including antibodies, agonists, and/orantagonists thereof) may be used in detecting, diagnosing, preventing,and/or treating.

Table 1E describes the use of FMAT technology, inter alia, for testingor demonstrating various biological activities. Fluorometric microvolumeassay technology (FMAT) is a fluorescence-based system which provides ameans to perform nonradioactive cell- and bead-based assays to detectactivation of cell signal transduction pathways. This technology wasdesigned specifically for ligand binding and immunological assays. Usingthis technology, fluorescent cells or beads at the bottom of the wellare detected as localized areas of concentrated fluorescence using adata processing system. Unbound flurophore comprising the backgroundsignal is ignored, allowing for a wide variety of homogeneous assays.FMAT technology may be used for peptide ligand binding assays,immunofluorescence, apoptosis, cytotoxicity, and bead-basedimmunocapture assays. See, Miraglia S et. al., “Homogeneous cell andbead based assays for highthroughput screening using flourometricmicrovolume assay technology,” Journal of Biomolecular Screening;4:193-204 (1999). In particular, FMAT technology may be used to test,confirm, and/or identify the ability of polypeptides (includingpolypeptide fragments and variants) to activate signal transductionpathways. For example, FMAT technology may be used to test, confirm,and/or identify the ability of polypeptides to upregulate production ofimmunomodulatory proteins (such as, for example, interleukins, GM-CSF,Rantes, and Tumor Necrosis factors, as well as other cellular regulators(e.g. insulin)).

Table 1E also describes the use of kinase assays for testing,demonstrating, or quantifying biological activity. In this regard, thephosphorylation and de-phosphorylation of specific amino acid residues(e.g. Tyrosine, Serine, Threonine) on cell-signal transduction proteinsprovides a fast, reversible means for activation and de-activation ofcellular signal transduction pathways. Moreover, cell signaltransduction via phosphorylation/de-phosphorylation is crucial to theregulation of a wide variety of cellular processes (e.g. proliferation,differentiation, migration, apoptosis, etc.). Accordingly, kinase assaysprovide a powerful tool useful for testing, confirming, and/oridentifying polypeptides (including polypeptide fragments and variants)that mediate cell signal transduction events via proteinphosphorylation. See e.g., Forrer, P., Tamaskovic R., and Jaussi, R.“Enzyme-Linked Immunosorbent Assay for Measurement of JNK, ERK, and p38Kinase Activities” Biol. Chem. 379(8-9): 1101-1110 (1998).

Description of Table 2

Table 2 summarizes homology and features of some of the polypeptides ofthe invention. The first column provides a unique clone identifier,“Clone ID”, corresponding to a cDNA clone disclosed in Table 1A or 1B.The second column provides the unique contig identifier, “Contig ID:”corresponding to contigs in Table 1B and allowing for correlation withthe information in Table 1B. The third column provides the sequenceidentifier, “SEQ ID NO:X”, for the contig polynucleotide sequence. Thefourth column provides the analysis method by which thehomology/identity disclosed in the Table was determined. Comparisonswere made between polypeptides encoded by the polynucleotides of theinvention and either a non-redundant protein database (herein referredto as “NR”), or a database of protein families (herein referred to as“PFAM”) as further described below. The fifth column provides adescription of the PFAM/NR hit having a significant match to apolypeptide of the invention. Column six provides the accession numberof the PFAM/NR hit disclosed in the fifth column. Column seven,“Score/Percent Identity”, provides a quality score or the percentidentity, of the hit disclosed in columns five and six. Columns 8 and 9,“NT From” and “NT To” respectively, delineate the polynucleotides in“SEQ ID NO:X” that encode a polypeptide having a significant match tothe PFAM/NR database as disclosed in the fifth and sixth columns. Inspecific embodiments polypeptides of the invention comprise, oralternatively consist of, an amino acid sequence encoded by apolynucleotide in SEQ ID NO:X as delineated in columns 8 and 9, orfragments or variants thereof.Description of Table 3Table 3 provides polynucleotide sequences that may be disclaimedaccording to certain embodiments of the invention. The first columnprovides a unique clone identifier, “Clone ID”, for a cDNA clone relatedto contig sequences disclosed in Table 1B. The second column providesthe sequence identifier, “SEQ ID NO:X”, for contig sequences disclosedin Table 1A and/or 1B. The third column provides the unique contigidentifier, “Contig ID:”, for contigs disclosed in Table 1B. The fourthcolumn provides a unique integer ‘a’ where ‘a’ is any integer between 1and the final nucleotide minus 15 of SEQ ID NO:X, and the fifth columnprovides a unique integer ‘b’ where ‘b’ is any integer between 15 andthe final nucleotide of SEQ ID NO:X, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:X, and where bis greater than or equal to a+14. For each of the polynucleotides shownas SEQ ID NO:X, the uniquely defined integers can be substituted intothe general formula of a−b, and used to describe polynucleotides whichmay be preferably excluded from the invention. In certain embodiments,preferably excluded from the invention are at least one, two, three,four, five, ten, or more of the polynucleotide sequence(s) having theaccession number(s) disclosed in the sixth column of this Table(including for example, published sequence in connection with aparticular BAC clone). In further embodiments, preferably excluded fromthe invention are the specific polynucleotide sequence(s) contained inthe clones corresponding to at least one, two, three, four, five, ten,or more of the available material having the accession numbersidentified in the sixth column of this Table (including for example, theactual sequence contained in an identified BAC clone).Description of Table 4Table 4 provides a key to the tissue/cell source identifier codedisclosed in Table 1B, column 8. Column 1 provides the tissue/cellsource identifier code disclosed in Table 1B, Column 8. Columns 2-5provide a description of the tissue or cell source. Codes correspondingto diseased tissues are indicated in column 6 with the word “disease”.The use of the word “disease” in column 6 is non-limiting. The tissue orcell source may be specific (e.g. a neoplasm), or may bedisease-associated (e.g., a tissue sample from a normal portion of adiseased organ). Furthermore, tissues and/or cells lacking the “disease”designation may still be derived from sources directly or indirectlyinvolved in a disease state or disorder, and therefore may have afurther utility in that disease state or disorder. In numerous caseswhere the tissue/cell source is a library, column 7 identifies thevector used to generate the library.Description of Table 5Table 5 provides a key to the OMIM reference identification numbersdisclosed in Table 1B, column 10. OMIM reference identification numbers(Column 1) were derived from Online Mendelian Inheritance in Man (OnlineMendelian Inheritance in Man, OMIM. McKusick-Nathans Institute forGenetic Medicine, Johns Hopkins University (Baltimore, Md.) and NationalCenter for Biotechnology Information, National Library of Medicine,(Bethesda, Md.) 2000. World Wide Web URL:http://www.ncbi.nlm.nih.gov/omim/). Column 2 provides diseasesassociated with the cytologic band disclosed in Table 1B, column 9, asdetermined using the Morbid Map database.Definitions

The following definitions are provided to facilitate understanding ofcertain terms used throughout this specification.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.The term “isolated” does not refer to genomic or cDNA libraries, wholecell total or mRNA preparations, genomic DNA preparations (includingthose separated by electrophoresis and transferred onto blots), shearedwhole cell genomic DNA preparations or other compositions where the artdemonstrates no distinguishing features of the polynucleotide/sequencesof the present invention.

In the present invention, a “secreted” protein refers to those proteinscapable of being directed to the ER, secretory vesicles, or theextracellular space as a result of a signal sequence, as well as thoseproteins released into the extracellular space without necessarilycontaining a signal sequence. If the secreted protein is released intothe extracellular space, the secreted protein can undergo extracellularprocessing to produce a “mature” protein. Release into the extracellularspace can occur by many mechanisms, including exocytosis and proteolyticcleavage.

As used herein, a “polynucleotide” refers to a molecule having a nucleicacid sequence contained in SEQ ID NO:X (as described in column 5 ofTable 1A), or cDNA clone (as described in column 2 of Table 1A andcontained within a pool of plasmids deposited with the ATCC in ATCCDeposit No: Z). For example, the polynucleotide can contain thenucleotide sequence of the full length cDNA sequence, including the 5′and 3′ untranslated sequences, the coding region, with or without anatural or artificial signal sequence, the protein coding region, aswell as fragments, epitopes, domains, and variants of the nucleic acidsequence. Moreover, as used herein, a “polypeptide” refers to a moleculehaving an amino acid sequence encoded by a polynucleotide of theinvention as broadly defined (obviously excluding poly-Phenylalanine orpoly-Lysine peptide sequences which result from translation of a polyAtail of a sequence corresponding to a cDNA).

In the present invention, a representative plasmid containing thesequence of SEQ ID NO:X was deposited with the American Type CultureCollection (“ATCC”) and/or described in Table 1A. As shown in Table 1A,each cDNA is identified by a cDNA clone identifier and the ATCC DepositNumber (ATCC Deposit No: Z). Plasmids that were pooled and deposited asa single deposit have the same ATCC Deposit Number. The ATCC is locatedat 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCCdeposit was made pursuant to the terms of the Budapest Treaty on theinternational recognition of the deposit of microorganisms for purposesof patent procedure.

A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:X, or the complementthereof (e.g., the complement of any one, two, three, four, or more ofthe polynucleotide fragments described herein) and/or sequences of thecDNA contained in the deposited clone (e.g., the complement of any one,two, three, four, or more of the polynucleotide fragments describedherein). “Stringent hybridization conditions” refers to an overnightincubation at 42 degree C. in a solution comprising 50% formamide, 5×SSC(750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1× SSC atabout 65 degree C.

Also included within “polynucleotides” of the present invention arenucleic acid molecules that hybridize to the polynucleotides of thepresent invention at lower stringency hybridization conditions. Changesin the stringency of hybridization and signal detection are primarilyaccomplished through the manipulation of formamide concentration (lowerpercentages of formamide result in lowered stringency); salt conditions,or temperature. For example, lower stringency conditions include anovernight incubation at 37 degree C. in a solution comprising 6×SSPE(20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30%formamide, 100 μg/ml salmon sperm blocking DNA; followed by washes at 50degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lowerstringency, washes performed following stringent hybridization can bedone at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, a polynucleotide which hybridizes only to polyA+sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a complementary stretch of T (or U) residues, would notbe included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

The polynucleotides of the present invention can be composed of anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide may also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

In specific embodiments, the polynucleotides of the invention are atleast 15, at least 30, at least 50, at least 100, at least 125, at least500, or at least 1000 continuous nucleotides but are less than or equalto 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb,2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides ofthe invention comprise a portion of the coding sequences, as disclosedherein, but do not comprise all or a portion of any intron. In anotherembodiment, the polynucleotides comprising coding sequences do notcontain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ tothe gene of interest in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1genomic flanking gene(s).

“SEQ ID NO:X” refers to a polynucleotide sequence described in column 5of Table 1A, while “SEQ ID NO:Y” refers to a polypeptide sequencedescribed in column 10 of Table 1A. SEQ ID NO:X is identified by aninteger specified in column 6 of Table 1A. The polypeptide sequence SEQID NO:Y is a translated open reading frame (ORF) encoded bypolynucleotide SEQ ID NO:X. The polynucleotide sequences are shown inthe sequence listing immediately followed by all of the polypeptidesequences. Thus, a polypeptide sequence corresponding to polynucleotidesequence SEQ ID NO:2 is the first polypeptide sequence shown in thesequence listing. The second polypeptide sequence corresponds to thepolynucleotide sequence shown as SEQ ID NO:3, and so on.

The polypeptides of the present invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).

The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

The polypeptides may be in the form of the secreted protein, includingthe mature form, or may be a part of a larger protein, such as a fusionprotein (see below). It is often advantageous to include an additionalamino acid sequence which contains secretory or leader sequences,pro-sequences, sequences which aid in purification, such as multiplehistidine residues, or an additional sequence for stability duringrecombinant production.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, including the secretedpolypeptide, can be substantially purified using techniques describedherein or otherwise known in the art, such as, for example, by theone-step method described in Smith and Johnson, Gene 67:31-40 (1988).Polypeptides of the invention also can be purified from natural,synthetic or recombinant sources using techniques described herein orotherwise known in the art, such as, for example, antibodies of theinvention raised against the polypeptides of the present invention inmethods which are well known in the art.

By a polypeptide demonstrating a “functional activity” is meant, apolypeptide capable of displaying one or more known functionalactivities associated with a full-length (complete) protein of theinvention. Such functional activities include, but are not limited to,biological activity, antigenicity [ability to bind (or compete with apolypeptide for binding) to an anti-polypeptide antibody],immunogenicity (ability to generate antibody which binds to a specificpolypeptide of the invention), ability to form multimers withpolypeptides of the invention, and ability to bind to a receptor orligand for a polypeptide.

“A polypeptide having functional activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical to, anactivity of a polypeptide of the present invention, including matureforms, as measured in a particular assay, such as, for example, abiological assay, with or without dose dependency. In the case wheredose dependency does exist, it need not be identical to that of thepolypeptide, but rather substantially similar to the dose-dependence ina given activity as compared to the polypeptide of the present invention(i.e., the candidate polypeptide will exhibit greater activity or notmore than about 25-fold less and, preferably, not more than abouttenfold less activity, and most preferably, not more than aboutthree-fold less activity relative to the polypeptide of the presentinvention).

The functional activity of the polypeptides, and fragments, variantsderivatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length polypeptide of the present inventionfor binding to an antibody to the full length polypeptide, variousimmunoassays known in the art can be used, including but not limited to,competitive and non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

In another embodiment, where a ligand is identified, or the ability of apolypeptide fragment, variant or derivative of the invention tomultimerize is being evaluated, binding can be assayed, e.g., by meanswell-known in the art, such as, for example, reducing and non-reducinggel chromatography, protein affinity chromatography, and affinityblotting. See generally, Phizicky, E., et al., Microbiol. Rev. 59:94-123(1995). In another embodiment, physiological correlates polypeptide ofthe present invention binding to its substrates (signal transduction)can be assayed. In addition, assays described herein (see Examples) andotherwise known in the art may routinely be applied to measure theability of polypeptides of the present invention and fragments, variantsderivatives and analogs thereof to elicit polypeptide related biologicalactivity (either in vitro or in vivo). Other methods will be known tothe skilled artisan and are within the scope of the invention.

Polynucleotides and Polypeptides of the Invention

Features of Protein Encoded by Gene No: 1

The translation product of this gene shares sequence homology withseveral human sodium-dependent phosphate transporters (See e.g.,Genebank Acc. Nos. gi|2062692 and gi|450532), and a rabbit renalcortical Na/Pa-i-cotransporter which is thought to be important incellular metabolism and kidney function (See Genbank Accession No.gi|165690).

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence:

HELGGLLADFLLSRKILRLITIRKLFTAIGVLFPSVILVSLPWVRSSHSMTMTFLVLSSAISSFCESGALVNFLDIAPRYTGFLKGLLQVFAHIAGAISPTAAGFFISQDSEFGWRNVFLLSAAVNISGLVFYLIFGRAD VQDWAKEQTFTHL (SEQ ID NO:123);HELGGLLADFLLSRKILRLITI (SEQ ID NO:125); RKLFTAIGVLFPSVILVSL PWVRS (SEQID NO:126); SHSMTMTFLVLSSAISSFCESGAL (SEQ ID NO:127);VNFLDIAPRYTGFLKGLLQVFAH (SEQ ID NO:128; IAGAISPTAAGFFISQDSEF GWRN (SEQID NO:129); VFLLSAAVNISGLVFYLIFGRADVQDWA KEQTFTHL (SEQ ID NO:130);LMKNPAAVGEMAPAMCAKTCNSPLRKPVYRGAISKKLTRAPDSQKLLMAEDSTKKVMVMLWLDLTQGRDTRITDGKRTPMAVKSFLMVMS LR IFLERRKSASRPPSSC(SEQ ID NO:124); LMKNPAAVGEMAPAMCAKTCNSPL RKPV (SEQ ID NO:131);YRGAISKKLTRAPDSQKLLMAED STKKVMVM (SEQ ID NO:132); and/orLWLDLTQGRDTRITDGKRTPMAVKSFLMVMSLRIFLERR KSASRPPSSC (SEQ ID NO:133).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

This gene is expressed primarily in human adult small intestine.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, renal and gastrointestinaldisorders, or other disorders where ion homeostasis is aberrant.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the renalsystem, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g., renal,neural, gastrointestinal, and cancerous and wounded tissues) or bodilyfluids (e.g., lymph, bile, serum, plasma, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in small intestine combined with the homology toa family of Sodium-dependent phosphate transporters suggests thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the study, diagnosis, treatment, and/or prevention of renal andgastointestinal disorders and/or diseases. The protein can also be usedto determine biological activity, to raise antibodies, as tissuemarkers, to isolate cognate ligands or receptors, to identify agentsthat modulate their interactions and as nutritional supplements. It mayalso have a very wide range of biological activities. Typical of theseare cytokine, cell proliferation/differentiation modulating activity orinduction of other cytokines; immunostimulating/immunosuppressantactivities (e.g., for treating human immunodeficiency virus infection,cancer, autoimmune diseases and allergy); regulation of hematopoiesis(e.g., for treating anaemia or as adjunct to chemotherapy); stimulationor growth of bone, cartilage, tendons, ligaments and/or nerves (e.g.,for treating wounds, stimulation of follicle stimulating hormone (forcontrol of fertility); chemotactic and chemokinetic activities (e.g.,for treating infections, tumors); hemostatic or thrombolytic activity(e.g., for treating haemophilia, cardiac infarction etc.);anti-inflammatory activity (e.g., for treating septic shock, Crohn'sdisease); as antimicrobials; for treating psoriasis or otherhyperproliferative diseases; for regulation of metabolism, and behavior.Also contemplated is the use of the corresponding nucleic acid in genetherapy procedures. Protein, as well as, antibodies directed against theprotein may show utility as a tumor marker and/or immunotherapy targetsfor the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:11 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 666 of SEQID NO:11, b is an integer of 15 to 680, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:11, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 2

The translation product of this gene shares sequence homology withseveral members of a recently described subfamily of P-type ATPases,these ATPases are thought to play a general role in ATP-dependentaminophospholipid transport. Members of this subfamily include, thehuman FIC1 PROTEIN (see Genbank Accession No. gi|3628757) which isthought to play an essential role in enterohepatic circulation of bileacids, as it's deletion has been linked to Cholestasis, or impaired bileflow. Other members include, the putative E1-E2 ATPase from Musmusculus, which is thought to be important in phagocytosis, bloodclotting, and cellular metabolism (See Genbank Accession No. gi|2895095(AF011337)), and Bovine and Murine chromaffin granule ATPase II homologs(see Genbank Accession No. gi|4115341 and gi|1663648)

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: EYSTPDTVHLRKTILFSVKVPVLSEKMYCICPKSSVMFRARHCSCESVSSSYNCMSWLMKYTWHALTISMEXYKEMGSKPAELYHVKNE LTAAVTGDKELPSDLGT(SEQ ID NO:134); NQGSAEQQWAPLQAXKLERQ (SEQ ID NO:135);YSSAGFDPISLYXSIEIVKACQVYFINQDMQLYDEETDSQLQCRALNITEDLGQIQYIFSDKTGTLTENKMVFRRCTVSGVEYSHDANEGLLRDAQWSTRLAGSISISFSGLLTGPCCFDSAPCLCLKF (SEQ ID NO:137);YSSAGFDPISLYXSIEIVKAC QVYFI (SEQ ID NO:138); NQDMQLYDEETDSQ LQCRALNITEDL(SEQ ID NO:139); GQIQYIFSDKTGTLTENKMVFRRCTVSG (SEQ ID NO:140); VEYSHDANEGLLRDAQWSTRLAGSISIS (SEQ ID NO:141); FSGLLTGPCCFDSAPCLCL KF (SEQ IDNO:142); and/or IRHETLRNTDAXXG IVIYAGHETKALLNNSGPRY KRXSWRGR (SEQ IDNO:136). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

The gene encoding the disclosed cDNA is believed to reside on chromosome15. Accordingly, polynucleotides related to this invention are useful asa marker in linkage analysis for chromosome 15.

This gene is expressed primarily in human prostate.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, reproductive or cellularmetabolism disorders. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thereproductive system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., immune, reproductive, and cancerous and wounded tissues) orbodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell samples takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in prostate tissue combined with the homology tomembers of a conserved subfamily of P-type ATPases suggests thatpolynucleotides and polypeptides corresponding to this gene are usefulfor treatment and diagnosis of reproductive, metabolic, or haemopoieticdisorders, such as congenital afflictions or proliferative conditions,including cancers. The expression in the prostate tissue may indicatethe gene or its products can be used in disorders of the prostate,including inflammatory disorders, such as chronic prostatitis,granulomatous prostatitis and malacoplakia, prostatic hyperplasia andprostate neoplastic disorders, including adenocarcinoma, transitionalcell carcinomas, ductal carcinomas, squamous cell carcinomas, or ashormones or factors with systemic or reproductive functions. Protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:12 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 727 of SEQID NO:12, b is an integer of 15 to 741, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:12, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 3

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: HELGPVCLHAIMLAELIFLFRSLHGILASAGTIGAVAAWL(SEQ ID NO:143). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

This gene is expressed primarily in human testes.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, reproductive disorders,particularly of the testes. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe male reproductive system, expression of this gene at significantlyhigher or lower levels may be routinely detected in certain tissues orcell types (e.g., reproductive, testes, and cancerous and woundedtissues) or bodily fluids (e.g., lymph, seminal fluid, serum, plasma,urine, synovial fluid and spinal fluid) or another tissue or cell sampletaken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.

The tissue distribution in testes suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosis,treatment, and/or prevention of various reproductive or endocrinedisorders, such as male infertility and hormone imbalances. Similarly,the tissue distribution in testes indicates that the protein product ofthis clone is useful for the treatment and diagnosis of conditionsconcerning normal testicular function (e.g., endocrine function, spermmaturation), as well as cancer. Therefore, this gene product is usefulin the treatment of male infertility and/or impotence. This gene productis also useful in assays designed to identify binding agents, as suchagents (antagonists) are useful as male contraceptive agents.

The secreted protein may also be used as a contraceptive. Similarly, theprotein is believed to be useful in the treatment and/or diagnosis oftesticular cancer. The testes are also a site of active gene expressionof transcripts that may be expressed, particularly at low levels, inother tissues of the body. Therefore, this gene product may be expressedin other specific tissues or organs where it may play related functionalroles in other processes, such as hematopoiesis, inflammation, boneformation, and kidney function, to name a few possible targetindications. Protein, as well as, antibodies directed against theprotein may show utility as a tumor marker and/or immunotherapy targetsfor the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:13 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 605 of SEQID NO:13, b is an integer of 15 to 619, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:13, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 4

The translation product of this clone has been shown to have homology tothe Y isoform of the conserved ubiquitous human TPR motif which isthought to be important in maintaining cellular metabolism in additionto a possible connection in increasing an individual's susceptibility tomale infertility (See Genbank Accession No. gi|2580574 (AF000994)). Inaddition, the translation product of this clone shows homology to themurine male-specific histocompatibility antigen H-YDb (See GenbankAccession No. gnl|PID|e300472) from which is derived an epitope of thecomplex male-specific transplantation antigen H-Y.

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: DFGTXSDPKLFEMIKYCLLKILKQYQTLREALVAAGKEVIWHGRTNDEPAHYCSICEVEVFNLLFVTNESNTQKTYIVHCHDCARKTSKSLENFVVLEQYKMEDLIQVYDQFTLASPWPPMDQSAFTSSLLRPIKALGSGRAEQTSGDQLQKGATHSRASSLLRAAEMTRRPASREELPDPGLFCHSIKLLFVLL (SEQ ID NO:144);DFGTXSDPKLFEMIKYCLLKILKQYQ (SEQ ID NO:145); TLREALVAAGKEVIWHGRTNDEPAHYCS(SEQ ID NO:146); ICEVEVFNLLF VTNESNTQKTYIVHC (SEQ ID NO:147);HDCARKTSKSLENFVVLEQYKMED LIQVYD (SEQ ID NO:148);QFTLASPWPPMDQSAFTSSLLRPIKALGSG (SEQ ID NO:149);RAEQTSGDQLQKGATHSRASSLLRAAEMT (SEQ ID NO:150); and/orRRPASREELPDPGLFCHSIKLLFVLL (SEQ ID NO:151). Polynucleotides encodingthese polypeptides are also encompassed by the invention.

This gene is expressed primarily in activated human neutrophils andsynovium.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, immune or hematopoieiticdisorders, particularly inflammatory conditions, and/or autoimmunedisorders, such as arthritis. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe inflammatory and immune systems, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., immune, skeletal, and cancerous andwounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in neutrophils and synovium, in addition to thehomology of the translated product of this clone to a murinemale-specific histocompatibility antigen, suggests that polynucleotidesand polypeptides corresponding to this gene are useful for the study,diagnosis, and treatment of inflammatory and immune disorders. Moreover,the protein may be useful for the detection and/or treatment ofdisorders and conditions afflicting the skeletal system, in particularosteoporosis, bone cancer, connective tissue disorders, (e.g.,arthritis, trauma, tendonitis, chrondomalacia and inflammation). Theprotein is also useful in the diagnosis or treatment of variousautoimmune disorders such as rheumatoid arthritis, lupus, scleroderma,and dermatomyositis, dwarfism, spinal deformation, joint abnormalities,and chondrodysplasias (ie. spondyloepiphyseal dysplasia congenita,familial osteoarthritis, Atelosteogenesis type II, metaphysealchondrodysplasia type Schmid). Protein, as well as, antibodies directedagainst the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:14 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 597 of SEQID NO:14, b is an integer of 15 to 611, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:14, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 5

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: LPGNFRPPRVILTFQWRFYLSFRKL (SEQ IDNO:152); XIPPXXLPGNFRPPRVILTFQWRFYLSFRKL (SEQ ID NO:154); and/orYLLLPCGLLSFWMCGALVVSPFVQNGQGQRLREARSLCLLKGTTWIFLMLSLPHFLVQELKFSNNFFSTVVIFSTSGFLQPTLIFLKLSWKSTHL (SEQ ID NO:153).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

This gene is expressed primarily in anergic T-cells.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, immune or hematopoieticdisorders, particularly inflammatory or immunodeficiency conditions.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the immunesystem, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g.,immune, hematopoietic, and cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid, and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in T-cells suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the study andtreatment of immune defects. More specifically, the gene product may beinvolved in the regulation of cytokine production, antigen presentation,or other processes that may also suggest a usefulness in the treatmentof cancer (e.g., by boosting immune responses).

Since the gene is expressed in cells of lymphoid origin, the naturalgene product may be involved in immune functions. Therefore it may bealso used as an agent for immunological disorders including arthritis,asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoidarthritis, granulomatous disease, inflammatory bowel disease, sepsis,acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such asT-cell mediated cytotoxicity; immune reactions to transplanted organsand tissues, such as host-versus-graft and graft-versus-host diseases,or autoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, sclerodermaand tissues. In addition, this gene product may have commercial utilityin the expansion of stem cells and committed progenitors of variousblood lineages, and in the differentiation and/or proliferation ofvarious cell types. Protein, as well as, antibodies directed against theprotein may show utility as a tumor marker and/or immunotherapy targetsfor the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:15 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 571 of SEQID NO:15, b is an integer of 15 to 585, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:15, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 6

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: YMMVHCKYSVYNLLNKWIGFSIFPHWTWIDLEIGGLNLQVEIKGPNNCRVAGEG RYKCSKGGSR (SEQ ID NO:155). Polynucleotides encodingthese polypeptides are also encompassed by the invention.

This gene is expressed primarily in anergic T-cells.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, immune and hemopoietic disorders,particularly inflammatory or immunodeficiency conditions. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., immune, hematopoietic, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution in T-cells suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the study andtreatment of inflammation, and other immune and hematopoietic disorders.Specifically, the gene product may be involved in the regulation ofcytokine production, antigen presentation, or other processes that mayalso suggest a usefulness in the treatment of cancer (e.g., by boostingimmune responses). Since the gene is expressed in cells of lymphoidorigin, the natural gene product may be involved in immune functions.Therefore it may be also used as an agent for immunological disordersincluding arthritis, asthma, immunodeficiency diseases such as AIDS,leukemia, rheumatoid arthritis, granulomatous disease, inflammatorybowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis,hypersensitivities, such as T-cell mediated cytotoxicity; immunereactions to transplanted organs and tissues, such as host-versus-graftand graft-versus-host diseases, or autoimmunity disorders, such asautoimmune infertility, lense tissue injury, demyelination, systemiclupus erythematosis, drug induced hemolytic anemia, rheumatoidarthritis, Sjogren's disease, scleroderma and tissues. In addition, thisgene product may have commercial utility in the expansion of stem cellsand committed progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:16 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 1026 of SEQID NO:16, b is an integer of 15 to 1040, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:16, and whereb is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 7

When tested against U937 Myeloid cell lines, supernatants removed fromcells containing this gene activated the GAS assay. Thus, it is likelythat this gene activates myeloid cells through the Jak-STAT signaltransduction pathway. The gamma activating sequence (GAS) is a promoterelement found upstream of many genes which are involved in the Jak-STATpathway. The Jak-STAT pathway is a large, signal transduction pathwayinvolved in the differentiation and proliferation of cells. Therefore,activation of the Jak-STAT pathway, reflected by the binding of the GASelement, can be used to indicate proteins involved in the proliferationand differentiation of cells.

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: MSAALWTYMRFLACLNHSSGSMYLSVNSTPVLLLLLVPNSARARAEFLQPGGXTSSRAAXXAVELQLLFPLXXG (SEQ ID NO:156);FRQARNLMYVHNAADIHSSLPQHITVISPRELCHTFSLLKPATLDLLCSLSVGN LFRISERQCKH (SEQID NO:157); and/or RVNVSSIMDIHEVPGLSKSQLWFNVPVCQLHTCVAVAARAEFGTSSCRIPAARGXH (SEQ ID NO:158). Polynucleotidesencoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in prostate cancer.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, reproductive disorders,particular prostate cancer or infertility. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the prostate, especially of the reproductive system,expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types (e.g., reproductive,prostate, and cancerous and wounded tissues) or bodily fluids (e.g.,lymph, seminal fluid, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in prostate cancer, along with activation of theGAS assay when supernatants of cells containing this gene were testedagainst U937 Myeloid cell lines, suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the treatment anddiagnosis of disorders of the reproductive system, particularproliferative conditions such as cancer. Similarly, the gene or itsproducts can be used in the disorders of the prostate, includinginflammatory disorders, such as chronic prostatitis, granulomatousprostatitis and malacoplakia, prostatic hyperplasia and prostateneoplastic disorders, including adenocarcinoma, transitional cellcarcinomas, ductal carcinomas, squamous cell carcinomas, or as hormonesor factors with systemic or reproductive functions. Protein, as well as,antibodies directed against the protein may show utility as atissue-specific marker and/or immunotherapy target for the above listedtissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:17 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 611 of SEQID NO:17, b is an integer of 15 to 625, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:17, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 8

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: IRHEGNSCTNKTAHAVLTASYTECSC (SEQ IDNO:159). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

This gene is expressed primarily in prostate.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, disorders of the reproductivesystem, particularly cancer. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe reproductive system, expression of this gene at significantly higheror lower levels may be routinely detected in certain tissues or celltypes (e.g., reproductive, prostate, and cancerous and wounded tissues)or bodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in prostate suggests that polynucleotides andpolypeptides corresponding to this gene are useful for treatment anddiagnosis of disorders of the reproductive system, such as proliferativeconditions, including, but not limited to, prostate cancer. Similarly,the gene or its products can be used in the treatment of disorders ofthe prostate, including inflammatory disorders, such as chronicprostatitis, granulomatous prostatitis and malacoplakia, prostatichyperplasia and prostate neoplastic disorders, including adenocarcinoma,transitional cell carcinomas, ductal carcinomas, squamous cellcarcinomas, or as hormones or factors with systemic or reproductivefunctions. Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:18 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 805 of SEQID NO:18, b is an integer of 15 to 819, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:18, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 9

This gene is expressed primarily in anergic T-cells.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, immune and blood disorders,particularly inflammatory or immunodeficiency conditions. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., immune, hematopoeitic, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution in T-cells suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the study andtreatment of immune and inflammatory disorders. Moreover, the geneproduct may be involved in the regulation of cytokine production,antigen presentation, or other processes that may also suggest ausefulness in the treatment of cancer (e.g., by boosting immuneresponses).

Since the gene is expressed in cells of lymphoid origin, the naturalgene product may be involved in immune functions. Therefore it may bealso used as an agent for immunological disorders including arthritis,asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoidarthritis, granulomatous disease, inflammatory bowel disease, sepsis,acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such asT-cell mediated cytotoxicity; immune reactions to transplanted organsand tissues, such as host-versus-graft and graft-versus-host diseases,or autoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, sclerodermaand tissues. In addition, this gene product may have commercial utilityin the expansion of stem cells and committed progenitors of variousblood lineages, and in the differentiation and/or proliferation ofvarious cell types. Protein, as well as, antibodies directed against theprotein may show utility as a tumor marker and/or immunotherapy targetsfor the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:19 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 768 of SEQID NO:19, b is an integer of 15 to 782, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:19, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 10

This gene is expressed primarily in anergic T-cells.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, immune or hematopoieticdisorders, particularly inflammatory conditions. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., immune, hematopoietic, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution in T-cells suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the study andtreatment of immune and blood disorders. Moreover, this gene product maybe involved in the regulation of cytokine production, antigenpresentation, or other processes that may also suggest a usefulness inthe treatment of cancer (e.g., by boosting immune responses). Since thegene is expressed in cells of lymphoid origin, the natural gene productmay be involved in immune functions. Therefore it may be also used as anagent for immunological disorders including arthritis, asthma,immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis,granulomatous disease, inflammatory bowel disease, sepsis, acne,neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cellmediated cytotoxicity, immune reactions to transplanted organs andtissues, such as host-versus-graft and graft-versus-host diseases, orautoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, sclerodermaand tissues.

In addition, this gene product may have commercial utility in theexpansion of stem cells and committed progenitors of various bloodlineages, and in the differentiation and/or proliferation of variouscell types. Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:20 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 641 of SEQID NO:20, b is an integer of 15 to 655, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:20, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 11

When tested against K562 cell lines, supernatants removed from cellscontaining this gene activated the ISRE (interferon-sensitive responsiveelement) pathway. Thus, it is likely that this gene activates leukemiacells, and to a lesser extent, in immune and hematopoietic cells andtissues, through the Jak-STAT signal transduction pathway. ISRE is apromoter element found upstream in many genes which are involved in theJak-STAT pathway. The Jak-STAT pathway is a large, signal transductionpathway involved in the differentiation and proliferation of cells.Therefore, activation of the Jak-STAT pathway, reflected by the bindingof the ISRE element, can be used to indicate proteins involved in theproliferation and differentiation of cells.

This gene is expressed primarily in smooth muscle.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, vascular disorders, particularlymicrovascular disease, atherosclerosis, aneurysm, and stroke. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the vascular and smooth muscletissues, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g,vascular tissue, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder.

The tissue distribution in smooth muscle, combined with the detectedISRE biological activity, suggests that polynucleotides and polypeptidescorresponding to this gene are useful for the study, diagnosis, andtreatment of vascular disorders. The protein product of this gene isuseful for the diagnosis, treatment, and/or prevention of conditions andpathologies of the cardiovascular system, such as heart disease,restenosis, atherosclerosis, stoke, angina, thrombosis, and woundhealing. Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:21 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 784 of SEQID NO:21, b is an integer of 15 to 798, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:21, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 12

The translation product of this gene shares sequence homology with thehuman IL-8 receptor (PF4AR), which is thought to be important ininflammatory disorders.

This gene is expressed primarily in synovial sarcoma.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, inflammatory disorders such asrheumatoid arthritis. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,immune, skeletal, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder.

The tissue distribution in synovial sarcoma, combined with the homologyto an IL-8 receptor (PF4AR), suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosis,treatment, and/or prevention of inflammatory disorders such asrheumatoid arthritis. Similarly, this gene product may also be usefulfor the detection and treatment of osteoporosis, bone cancer, as wellas, disorders afflicting connective tissues (e.g., arthritis, trauma,tendonitis, chrondomalacia and inflammation), such as in the diagnosisor treatment of various autoimmune disorders such as rheumatoidarthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism,spinal deformation, and specific joint abnormalities as well aschondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familialosteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasiatype Schmid).

The secreted protein can also be used to determine biological activity,to raise antibodies, as tissue markers, to isolate cognate ligands orreceptors, to identify agents that modulate their interactions, and asnutritional supplements. It may also have a very wide range ofbiological activities. Typical of these are cytokine, cellproliferation/differentiation modulating activity or induction of othercytokines; immunostimulating/immunosuppressant activities (e.g. fortreating human immunodeficiency virus infection, cancer, autoimmunediseases and allergy); regulation of hematopoiesis (e.g. for treatinganemia or as adjunct to chemotherapy); stimulation or growth of bone,cartilage, tendons, ligaments and/or nerves (e.g. for treating wounds,stimulation of follicle stimulating hormone (for control of fertility);chemotactic and chemokinetic activities (e.g. for treating infections,tumors); hemostatic or thrombolytic activity (e.g. for treatinghemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g.for treating septic shock, Crohn's disease); as antimicrobials; fortreating psoriasis or other hyperproliferative diseases; for regulationof metabolism, and behavior. Also contemplated is the use of thecorresponding nucleic acid in gene therapy procedures. Protein, as wellas, antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:22 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 632 of SEQID NO:22, b is an integer of 15 to 646, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:22, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 13

When tested against PC12 cell lines, supernatants removed from cellscontaining this gene activated the EGR1 (early growth response gene 1)promoter element. Thus, it is likely that this gene activates sensoryneuron cells, and to a lesser extent, in neural cells and tissues,through the EGR1 signal transduction pathway. EGR1 is a separate signaltransduction pathway from Jak-STAT. Genes containing the EGR1 promoterelement are induced in various tissues and cell types upon activation,leading the cells to undergo differentiation and proliferation.

This gene is expressed primarily in human testes.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, reproductive disorders,particularly afflictions of the testes, such as male infertility andtesticular cancer. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thereproductive system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., reproductive, testes, and cancerous and wounded tissues) orbodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in testes combined with the detected EGR1biological activity suggests that polynucleotides and polypeptidescorresponding to this gene are useful for various reproductive disorderssuch as male infertility or associated endocrine disorders. Similarly,the protein product of this clone is useful for the treatment anddiagnosis of conditions concerning proper testicular function (e.g.,endocrine function, sperm maturation), as well as cancer. Therefore,this gene product is useful in the treatment of male infertility and/orimpotence. This gene product is also useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. Similarly, the protein is believed to be useful inthe treatment and/or diagnosis of testicular cancer. The testes are alsoa site of active gene expression of transcripts that may be expressed,particularly at low levels, in other tissues of the body. Therefore,this gene product may be expressed in other specific tissues or organswhere it may play related functional roles in other processes, such ashematopoiesis, inflammation, bone formation, and kidney function, toname a few possible target indications. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:23 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 738 of SEQID NO:23, b is an integer of 15 to 752, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:23, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 14

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence:YKVVLVWREDQSSHKIHLSQTLIQNKALTLFNSMKAERGEEAXGKNVSS (SEQ ID NO:160);YKVVLVWREDQSSHKIHLSQTLIQ (SEQ ID NO:162); NKALTLFNSMKAERGEEAXGKNVSS (SEQID NO:163); DGELSKCCMCSDYTIDCYFPISLPLLGRPYYLRHNIEIRPYINHTMMASKGSSKRMGCTSFTLTQKLEIIILSEKGMWKAEIGQKLGXLHHS (SEQ ID NO:161); DGELSKCCMCSDYTIDCYFPISLPLLGRPYY (SEQ ID NO:164); LRHNIEIRPYINHTMASKGSSKRMGCT SFTLT (SEQID NO:165); and/or QKLEIIILSEKGMWKAEIGQKLGXLHHS (SEQ ID NO:166).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

This gene is expressed primarily in fetal bone and testical tumors.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, developmental, skeletal, orreproductive disorders, particularly, proliferative conditions such astesticular cancer. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of themale reproductive systems, expression of this gene at significantlyhigher or lower levels may be routinely detected in certain tissues orcell types (e.g., reproductive, skeletal, testes, and cancerous andwounded tissues) or bodily fluids (e.g., lymph, seminal fluid, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution in testes suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosis,treatment, and/or prevention of testicular cancer. In addition,expression of this gene product in the testis may implicate this geneproduct in normal testicular function. In addition, this gene productmay be useful in the treatment of male infertility, and/or could be usedas a male contraceptive.

Moreover, this gene product may be useful in the detection and treatmentof disorders and conditions afflicting the skeletal system, inparticular osteoporosis, bone cancer, as well as, disorders afflictingconnective tissues (e.g., arthritis, trauma, tendonitis, chrondomalaciaand inflammation), such as in the diagnosis or treatment of variousautoimmune disorders such as rheumatoid arthritis, lupus, scleroderma,and dermatomyositis as well as dwarfism, spinal deformation, andspecific joint abnormalities as well as chondrodysplasias (ie.spondyloepiphysial dysplasia congenita, familial osteoarthritis,Atelosteogenesis type II, metaphysial chondrodysplasia type Schmid).

Expression within fetal tissue and other cellular sources marked byproliferating cells suggests that this protein may play a role in theregulation of cellular division, and may show utility in the diagnosis,treatment, and/or prevention of cancer and other proliferativedisorders. Similarly, developmental tissues rely on decisions involvingcell differentiation and/or apoptosis in pattern formation. Thus, thisprotein may also be involved in apoptosis or tissue differentiation andcould again be useful in cancer therapy. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:24 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 801 of SEQID NO:24, b is an integer of 15 to 815, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:24, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 15

This gene is expressed primarily in stomach.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, gastrointestinal and digestivedisorders. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thegastrointestinal system, expression of this gene at significantly higheror lower levels may be routinely detected in certain tissues or celltypes (e.g., gastrointestinal, stomach, and cancerous and woundedtissues) or bodily fluids (e.g., lymph, bile, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in stomach tissue suggests that polynucleotidesand polypeptides corresponding to this gene are useful for the study andtreatment of gastrointestinal, digestive, and general metabolicdisorders. For example, the protein product of this clone may be usefulfor the diagnosis, prevention, and/or treatment of various metabolicdisorders such as Tay-Sach's disease, phenylkenonuria, galactosemia,porphyrias, and Hurler's syndrome. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:25 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 864 of SEQID NO:25, b is an integer of 15 to 878, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:25, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 16

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: MLCINVQTHVYECA (SEQ ID NOP:167).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

This gene is expressed primarily in anergic T-cells and CD34 depletedcord blood.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, hemopoietic, immune, orreproductive conditions. Similarly, polypeptides and antibodies directedto these polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,hemopoietic, immune, or reproductive, and cancerous and wounded tissues)or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in T-cells suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the study andtreatment of immune, inflammatory and other hematopoietic disorders. Inaddition, polynucleotides and polypeptides corresponding to this geneare useful for the treatment and diagnosis of hematopoetic relateddisorders such as anemia, pancytopenia, leukopenia, thrombocytopenia orleukemia since stromal cells are important in the production of cells ofhematopoietic lineages.

The uses include bone marrow cell ex vivo culture, bone marrowtransplantation, bone marrow reconstitution, radiotherapy orchemotherapy of neoplasia. The gene product may also be involved inlymphopoiesis, therefore, it can be used in immune disorders such asinfection, inflammation, allergy, immunodeficiency etc. In addition,this gene product may have commercial utility in the expansion of stemcells and committed progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:26 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 836 of SEQID NO:26, b is an integer of 15 to 850, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:26, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 17

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: LCCPGWSAVVRSWLTATLASWVQAILMDSASQVAGITSVHHQAQLSFVFLVEMGLCHVGQAGLKLLASSDLPASASQSAGITGMSHHS WPERTSFIFKI (SEQ IDNO:168); LCCPGWSAVVRSWLTATLASWVQAILMD SASQ (SEQ ID NO:169);VAGITSVHHQAQLSFVFLVEMGLCHVGQAGLKLLA (SEQ ID NO:170); and/orSSDLPASASQSAGITGMSHHSWPERTSFIFKI (SEQ ID NO:171). Polynucleotidesencoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in human adult small intestine.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, gastrointestinal disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of thegastrointestinal system, expression of this gene at significantly higheror lower levels may be routinely detected in certain tissues or celltypes (e.g., gastrointestinal, metabolic, and cancerous and woundedtissues) or bodily fluids (e.g., bile, lymph, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in small intestine suggests that polynucleotidesand polypeptides corresponding to this gene are useful for the study,diagnosis, and treatment of gastrointestinal disorders, and/or treatmentof various metabolic disorders such as Tay-Sachs disease,phenylkenonuria, galactosemia, porphyrias, and Hurler's syndrome.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:27 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 774 of SEQID NO:27, b is an integer of 15 to 788, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:27, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 18

The translation product of this gene was shown to have homology to theF33H2.2 protein from Caenorhabditis elegans (See Genbank Accession No.gnl|PID|e1297838). Considering the homology to a C. elegans protein, animportant and vital function may be attributed to this clone based uponits conservation.

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: FGRGNTILFLRHNKDLVAQTAQPDQPNYGFPLDLLRCESLLGLDPATCSRVLNKNYTLLVSMAPLTNEIRPVSSCTPQHIGPAIPEVSSVWFKLYIYHVTGQGPPSLLLSKGTRLRKLPDIFQSYDRLXITSWGHDPGVVPTSNVLTMLNDALTHSAVLIQGHGLHGIGETVHVPFPFDETELQGEFTRVNMGVHKALQILRNRVXLQHLCGYVTMLNASSQLADRKLSDASDERGEPDLASGSDVNGSTESFEMVIEEATIDSATKQTSGATTEADWVP LV (SEQ ID NO:172); FGRGNTILFLRHNKDLVAQTAQPDQPNYGFPLDLLRCESLLGLDPATCSRVLNKNYTLLVSMAPLTNEIRPVSSCTPQHIGPAIPEVSSVWFKLYIYHVTGQGPPSLLLSKGTRLRKLPDIFQSYDRLXITSWGHDPGVVPTSNVLTMLNDALTHSAVLIQGHGLH GIGETVHVP (SEQ IDNO:173); LRHNKDLVAQTAQPDQPNYGF(SEQ ID NO: 174); FPLDLLRCESLLGLDPATCSR(SEQ ID NO: 175); RVLNKNYTLL VSMAPLTNEIR (SEQ ID NO: 176); RPVSSCTPQHIGPAIPEVSS (SEQ ID NO: 177); SVWFKLYIYHVTGQGPPSLLL (SEQ ID NO:178); LSKGTRLRKLPDIF QSYDRLX (SEQ ID NO: 179); XITSWGHDPGVV PTSNVLTM(SEQ ID NO: 180); MLNDALTHSAVLIQGHGLHGI(SEQ ID NO: 181); FPFDETELQGEFTRVNMGVHKALQILRNRVXLQHLCGYVTMLNASSQLADRKLSDASDERGEPDLASGSDVNGSTESFEMVIEEATIDSATKQTSGATTEADWVP LV (SEQ ID NO: 182);GEFTRVNMGVHKALQILRNRV (SEQ ID NO: 183); VXLQHLCGYVTML NASSQLA (SEQ IDNO: 184); ADRKLSDASDERGEPDLASGS (SEQ ID NO: 185); LRKLHSQTNPI (SEQ IDNO: 187); and/or SDVNGSTESFEMVIEEATIDS (SEQ ID NO: 186). Polynucleotidesencoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in healing groin wound, and to a lesserextent, in synovium.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, developmental, wound healing, andsynovial disorders. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thesynovium and epithelium, expression of this gene at significantly higheror lower levels may be routinely detected in certain tissues or celltypes (e.g., developmental, skeletal, and cancerous and wounded tissues)or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in wounded tissue suggests that polynucleotidesand polypeptides corresponding to this gene are useful for the study,diagnosis, and treatment of disorders involving the synovium andepithelium. Specifically, polynucleotides and polypeptides correspondingto this gene are useful for the treatment, diagnosis, and/or preventionof various skin disorders including congenital disorders (i.e. nevi,moles, freckles, Mongolian spots, hemangiomas, port-wine syndrome),integumentary tumors (i.e. keratoses, Bowen's disease, basal cellcarcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease,mycosis fungoides, and Kaposi's sarcoma), injuries and inflammation ofthe skin (i.e. wounds, rashes, prickly heat disorder, psoriasis,dermatitis), atherosclerosis, uticaria, eczema, photosensitivity,autoimmune disorders (i.e. lupus erythematosus, vitiligo,dermatomyositis, morphea, scleroderma, pemphigoid, and pemphigus),keloids, striae, erythema, petechiae, purpura, and xanthelasma. Inaddition, such disorders may predispose an individual viral andbacterial infections of the skin (i.e. cold sores, warts, chickenpox,molluscum contagiosum, herpes zoster, boils, cellulitis, erysipelas,impetigo, tinea, althletes foot, and ringworm).

Moreover, the protein product of this clone may also be useful for thetreatment or diagnosis of various connective tissue disorders such asarthritis, trauma, tendonitis, chrondomalacia and inflammation,autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma,and dermatomyositis as well as dwarfism, spinal deformation, andspecific joint abnormalities as well as chondrodysplasias (ie.spondyloepiphyseal dysplasia congenita, familial osteoarthritis,Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Theprotein is useful in the detection, treatment, and/or prevention ofskeletal diseases and disorders, which include, but are not limited toosteoporosis, bone cancer, etc. Protein, as well as, antibodies directedagainst the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:28 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 824 of SEQID NO:28, b is an integer of 15 to 838, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:28, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 19

This gene is expressed primarily in ovarian cancer.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, disorders of the reproductivesystem, particularly proliferative disorders of the ovary, such ascancer and cysts. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thereproductive system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., reproductive, ovarian tissue, and cancerous and wounded tissues)or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in ovarian tissue suggests that polynucleotidesand polypeptides corresponding to this gene are useful for the treatmentand diagnosis of disorders of the reproductive system and cancers.Moreover, the expression within cellular sources marked by proliferatingcells (i.e., ovarian cancer tissues) suggests this protein may play arole in the regulation of cellular division, and may show utility in thediagnosis, treatment, and/or prevention of developmental diseases anddisorders, cancer, and other proliferative conditions. Similarly,developmental tissues rely on decisions involving cell differentiationand/or apoptosis in pattern formation.

Dysregulation of apoptosis can result in inappropriate suppression ofcell death, as occurs in the development of some cancers, or in failureto control the extent of cell death, as is believed to occur in acquiredimmunodeficiency and certain neurodegenerative disorders, such as spinalmuscular atrophy (SMA). Therefore, the polynucleotides and polypeptidesof the present invention are useful in treating, detecting, and/orpreventing said disorders and conditions, in addition to other types ofdegenerative conditions. Thus this protein may modulate apoptosis ortissue differentiation and would be useful in the detection, treatment,and/or prevention of degenerative or proliferative conditions anddiseases. The protein is useful in modulating the immune response toaberrant polypeptides, as may exist in proliferating and cancerous cellsand tissues. The protein can also be used to gain new insight into theregulation of cellular growth and proliferation. Protein, as well as,antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:29 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 741 of SEQID NO:29, b is an integer of 15 to 755, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:29, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 20

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: RNFYLYFLPYCVVCVC (SEQ ID NO:188).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

This gene is expressed primarily in prostate cancer.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, reproductive disorders,particularly proliferative conditions of the prostate, such as cancer.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of thereproductive disorders and cancer, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., reproductive, prostate, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, amnioticfluid, seminal fluid, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in prostate suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the treatment anddiagnosis of disorders of the reproductive organs and cancers, such asmale infertility. Protein may also be useful as a contraceptive.Moreover, the expression within cellular sources marked by proliferatingcells (i.e., prostate cancer) suggests this protein may play a role inthe regulation of cellular division, and may show utility in thediagnosis, treatment, and/or prevention of developmental diseases anddisorders, cancer, and other proliferative conditions. Similarly,developmental tissues rely on decisions involving cell differentiationand/or apoptosis in pattern formation.

Dysregulation of apoptosis can result in inappropriate suppression ofcell death, as occurs in the development of some cancers, or in failureto control the extent of cell death, as is believed to occur in acquiredimmunodeficiency and certain neurodegenerative disorders, such as spinalmuscular atrophy (SMA). Therefore, the polynucleotides and polypeptidesof the present invention are useful in treating, detecting, and/orpreventing said disorders and conditions, in addition to other types ofdegenerative conditions. Thus this protein may modulate apoptosis ortissue differentiation and would be useful in the detection, treatment,and/or prevention of degenerative or proliferative conditions anddiseases. The protein is useful in modulating the immune response toaberrant polypeptides, as may exist in proliferating and cancerous cellsand tissues. The protein can also be used to gain new insight into theregulation of cellular growth and proliferation. Protein, as well as,antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues. Theprotein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:30 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 799 of SEQID NO:30, b is an integer of 15 to 813, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:30, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 21

This gene is expressed primarily in B-cells and rhabdomyosarcoma.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, immune, hematopoietic, ormuscular disorders, particularly proliferative conditions such as canceror fibroids. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thehaemopoietic and muscular system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., immune, hematopoietic, muscular,and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution in immune and muscle tissues or cells suggeststhat polynucleotides and polypeptides corresponding to this gene areuseful for the treatment and diagnosis of a variety of disorders, suchas for the detection, and/or prevention of muscular dystrophy,cardiomyopathy, fibroids, myomas, and rhabdomyosarcomas. This geneproduct may be involved in the regulation of cytokine production,antigen presentation, or other processes suggesting a usefulness in thetreatment of cancer (e.g., by boosting immune responses). Since the geneis expressed in cells of lymphoid origin, the natural gene product maybe involved in immune functions. Therefore it may be also used as anagent for immunological disorders including arthritis, asthma,immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis,granulomatous disease, inflammatory bowel disease, sepsis, acne,neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cellmediated cytotoxicity; immune reactions to transplanted organs andtissues, such as host-versus-graft and graft-versus-host diseases, orautoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, sclerodermaand tissues.

Moreover, the protein may represent a secreted factor that influencesthe differentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. In addition, this gene productmay have commercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:31 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 499 of SEQID NO:31, b is an integer of 15 to 513, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:3 1, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 22

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: GTRSINLLFFRCILEGGKSVEEQLCNSYKFS (SEQ IDNO:189). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

This gene is expressed primarily in anergic T-cells.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, immune and blood conditions,particularly inflammatory or immunodeficiency disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., immune, hematopoietic, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution in T-cells suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the study andtreatment of inflammation and immune disorders. More specifically, thegene product may be involved in the regulation of cytokine production,antigen presentation, or other processes that may also suggest ausefulness in the treatment of cancer (e.g., by boosting immuneresponses). Since the gene is expressed in cells of lymphoid origin, thenatural gene product may be involved in immune functions. Therefore itmay be also used as an agent for immunological disorders includingarthritis, asthma, immunodeficiency diseases such as AIDS, leukemia,rheumatoid arthritis, granulomatous disease, inflammatory bowel disease,sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities,such as T-cell mediated cytotoxicity; immune reactions to transplantedorgans and tissues, such as host-versus-graft and graft-versus-hostdiseases, or autoimmunity disorders, such as autoimmune infertility,lense tissue injury, demyelination, systemic lupus erythematosis, druginduced hemolytic anemia, rheumatoid arthritis, Sjogren's disease,scleroderma and tissues. In addition, this gene product may havecommercial utility in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:32 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 562 of SEQID NO:32, b is an integer of 15 to 576, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:32, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 23

The translation product of this gene was shown to have homology to thegi|2501808 brain digoxin carrier protein of Rattus norvegicus which isthought to serve the role as a sodium-independent organic aniontransporter. This gene may also play a role in hormone transport. Whentested against U937 cell lines, supernatants removed from cellscontaining this gene activated the GAS (gamma activating sequence)promoter element. Thus, it is likely that this gene activatespromyelocytic cells, and to a lesser extent in other immune orhematopoietic cells and tissues, through the Jak-STAT signaltransduction pathway. GAS is a promoter element found upstream of manygenes which are involved in the Jak-STAT pathway. The Jak-STAT pathwayis a large, signal transduction pathway involved in the differentiationand proliferation of cells. Therefore, activation of the Jak-STATpathway, reflected by the binding of the GAS element, can be used toindicate proteins involved in the proliferation and differentiation ofcells.

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: LTVPRRCPAATETNVDGQKVYRDCSCIPQNLSSGFGHATAGXMHFNLSEKAPPSGFHIRCEFSLHSXSSIPALTATLRCVRDPQRSFALGIQWIVVRILGGIPGPIAFGWVIDKACLLWQXQCGQXGSCLVYQXRP (SEQ ID NO:190);VSLCHAGALQPRRR (SEQ ID NO:191); ATETNVDGQKVYRDCSC IPQN (SEQ ID NO:192);NLSSGFGHATAGXMHFNLSEK (SEQ ID NO:193); KAPPSGFHIRCEFSLHSXSSI (SEQ IDNO:194); IPALTATLRCVRDPQRSFAL (SEQ ID NO: 195); and/orLGIQWIVVRILGGIPGPIAFG (SEQ ID NO:196). Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

This gene is expressed primarily in retina.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, visual disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the eye, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., opthalmic tissue, retinal tissue,cancerous and wounded tissues) or bodily fluids (e.g., lymph, vitreoushumor, aqueous humor, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in retinal tissue combined with the detected GASbiological activity suggests that polynucleotides and polypeptidescorresponding to this gene are useful for the diagnosis, treatment,and/or prevention of visual disorders of the eye. The protein is usefulin modulating the immune response to aberrant polypeptides, as may existin proliferating and cancerous cells and tissues. Protein, as well as,antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:33 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 1005 of SEQID NO:33, b is an integer of 15 to 1019, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:33, and whereb is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 24

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: GTAHLPTLHWKPLLS (SEQ ID NO:197).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

This gene is expressed primarily in synovial fluid of a patient withchronic synovitis.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, immune or skeletal disorders,particularly inflammatory disorders such as arthritis. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., immune, hematopoietic, skeletal,and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution in synovial fluid suggests that polynucleotidesand polypeptides corresponding to this gene are useful for thediagnosis, treatment, and/or prevention of inflammatory disorders suchas arthritis. Moreover, the expression of this gene product in synoviumwould suggest a role in the detection and treatment of disorders andconditions afflicting the skeletal system, in particular osteoporosis,bone cancer, connective tissue disorders (e.g., trauma, tendonitis,chrondomalacia and inflammation). The protein is also useful in thediagnosis or treatment of various autoimmune disorders (i.e., rheumatoidarthritis, lupus, scleroderma, and dermatomyositis), dwarfism, spinaldeformation, joint abnormalities, and chondrodysplasias (ie.spondyloepiphyseal dysplasia congenita, familial osteoarthritis,Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid).Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:34 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 419 of SEQID NO:34, b is an integer of 15 to 433, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:34, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 25

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: LVMQCLGQVLSPLRTSVCLPIERGRWPGMVPHTTSAL GG(SEQ ID NO:198); EPACLSH (SEQ ID NO:200); and/or QNTIHSLLPQGRMTKSLVLEEQKRKAGRSEMKLELLMRVSLWYSGQALVLLGLITNLSCSVLGKS FHLSGPLSVSL (SEQ IDNO:199). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

This gene is expressed primarily in human synovium.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, immune and skeletal diseasesand/or disorders, particularly inflammatory conditions, such asarthritis. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune and inflammatory systems, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., skeletal, synovium, and cancerousand wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,urine, synovial fluid and spinal fluid) or another tissue or cell sampletaken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.

The tissue distribution in synovium suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the study,diagnosis, and treatment of immune and inflammatory disorders. Inaddition, the gene product may play a role in the detection andtreatment of disorders and conditions affecting the skeletal system, inparticular osteoporosis, bone cancer, connective tissue disorders (e.g.,arthritis, trauma, tendonitis, chrondomalacia and inflammation). Theprotein is also useful in the diagnosis or treatment of autoimmunedisorders which include rheumatoid arthritis, lupus, scleroderma,dermatomyositis, dwarfism, spinal deformation, joint abnormalities, andchondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familialosteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasiatype Schmid). Protein, as well as, antibodies directed against theprotein may show utility as a tumor marker and/or immunotherapy targetsfor the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:35 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 628 of SEQID NO:35, b is an integer of 15 to 642, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:35, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 26

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: RDFGCEPSPGTDTGSLSFLV (SEQ ID NO:201).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

This gene is expressed primarily in synovial fluid of a patient withchronic synovitis.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, immune or skeletal disorders,particularly inflammatory disorders such as arthritis. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., immune, skeletal, and cancerousand wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,urine, synovial fluid and spinal fluid) or another tissue or cell sampletaken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.

The tissue distribution in synovium suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosis,treatment, and/or prevention of inflammatory disorders such asarthritis. In addition, the gene product may play a role in thedetection and treatment of disorders and conditions afflicting theskeletal system, in particular osteoporosis, bone cancer, as well as,disorders of the connective tissues (e.g., arthritis, trauma,tendonitis, chrondomalacia and inflammation). Moreover, the protein isalso useful in the diagnosis or treatment of various autoimmunedisorders such as rheumatoid arthritis, lupus, scleroderma,dermatomyositis, dwarfism, spinal deformation, and specific jointabnormalities as well as chondrodysplasias (ie. spondyloepiphysealdysplasia congenita, familial osteoarthritis, Atelosteogenesis type II,metaphyseal chondrodysplasia type Schmid). Protein, as well as,antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:36 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 653 of SEQID NO:36, b is an integer of 15 to 667, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:36, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 27

When tested against K562 cell lines, supernatants removed from cellscontaining this gene activated the ISRE (interferon-sensitive responsiveelement) promoter element. Thus, it is likely that this gene activatesleukemia cells through the Jak-STAT signal transduction pathway. ISRE isa promoter element found upstream in many genes which are involved inthe Jak-STAT pathway. The Jak-STAT pathway is a large, signaltransduction pathway involved in the differentiation and proliferationof cells. Therefore, activation of the Jak-STAT pathway, reflected bythe binding of the ISRE element, can be used to indicate proteinsinvolved in the proliferation and differentiation of cells.

This gene is expressed primarily in anergic T-cells.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, immune and blood disorders,particularly inflammatory or immunodeficiency disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., immune, hematopoietic, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution in T-cells, combined with the detected ISREbiological activity suggests that polynucleotides and polypeptidescorresponding to this gene are useful for the study and treatment ofimmune and blood diseases. More specifically, the gene product may beinvolved in the regulation of cytokine production, antigen presentation,or other processes that may also suggest a usefulness in the treatmentof cancer (e.g., by boosting immune responses). Since the gene isexpressed in cells of lymphoid origin, the natural gene product may beinvolved in immune functions. Therefore it may be also used as an agentfor immunological disorders including arthritis, asthma,immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis,granulomatous disease, inflammatory bowel disease, sepsis, acne,neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cellmediated cytotoxicity; immune reactions to transplanted organs andtissues, such as host-versus-graft and graft-versus-host diseases, orautoimmunity disorders, such as autoimmune infertility, lense tissueinjury, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjogren's disease, sclerodermaand tissues. In addition, this gene product may have commercial utilityin the expansion of stem cells and committed progenitors of variousblood lineages, and in the differentiation and/or proliferation ofvarious cell types. Protein, as well as, antibodies directed against theprotein may show utility as a tumor marker and/or immunotherapy targetsfor the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:37 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 640 of SEQID NO:37, b is an integer of 15 to 654, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:37, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 28

When tested against K562 cell lines, supernatants removed from cellscontaining this gene activated the ISRE (interferon-sensitive responsiveelement) promoter element. Thus, it is likely that this gene activatesleukemia cells, and to a lesser extent, in immune and hematopoeiticcells and tissues, through the Jak-STAT signal transduction pathway.ISRE is a promoter element found upstream in many genes which areinvolved in the Jak-STAT pathway. The Jak-STAT pathway is a large,signal transduction pathway involved in the differentiation andproliferation of cells. Therefore, activation of the Jak-STAT pathway,reflected by the binding of the ISRE element, can be used to indicateproteins involved in the proliferation and differentiation of cells.

This gene is expressed primarily in human fibrosarcoma.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, muscle disorders, particularlyproliferative conditions such as cancer or fibroids. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the metabolic system, expression ofthis gene at significantly higher or lower levels may be routinelydetected in certain tissues or cell types (e.g., muscle, and cancerousand wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,urine, synovial fluid and spinal fluid) or another tissue or cell sampletaken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.

The tissue distribution in fibrosarcoma, combined with the detected ISREbiological activity, suggests that polynucleotides and polypeptidescorresponding to this gene are useful for the study, diagnosis, and/ortreatment of various cancers. Protein, as well as, antibodies directedagainst the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:38 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 717 of SEQID NO:38, b is an integer of 15 to 731, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:38, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 29

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: SVILLCPFF (SEQ ID NO:202).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

This gene is expressed primarily in salivary gland.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, epithelial, immune, and digestivedisorders. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theepithelial, digestive, and immune systems, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., epithelial, immune, digestivetissues, and cancerous and wounded tissues) or bodily fluids (e.g.,lymph, serum, plasma, urine, synovial fluid and spinal fluid) or anothertissue or cell sample taken from an individual having such a disorder,relative to the standard gene expression level, i.e., the expressionlevel in healthy tissue or bodily fluid from an individual not havingthe disorder.

The tissue distribution in salivary tissue suggests that polynucleotidesand polypeptides corresponding to this gene are useful for the study,diagnosis, and treatment of various disorders of the immune, digestiveand epithelial systems. The protein is useful in modulating the immuneresponse to aberrant polypeptides, as may be present in the cells andtissues of proliferative organs. The protein may also be useful inenhancing or inhibiting the bodies antibiological and microbial defenses(i.e., by enhancing the secretion of an antibiological agent in saliva,such as nitric oxide, for example). Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:39 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 364 of SEQID NO:39, b is an integer of 15 to 378, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:39, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 30

This gene is expressed primarily in spongy brain tissue obtained fromAlzheimer's patients.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, Alzheimer's disease;neurodegenerative disorders including, but not restricted toAlzheimer's, such as schizophrenia, ALS, etc. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the brain and nervous system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., neural, cancerous and woundedtissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution suggests that polynucleotides and polypeptidescorresponding to this gene are useful for the diagnosis and/or treatmentof neurodegenerative disorders, particularly Alzheimer's disease.Specific expression of this gene product in the brain tissue ofAlzheimer's patients suggests that it may play a deleterious role in theprogression of the disease. Alternately, it may represent an attemptedresponse by the body to combat the progression of Alzheimer's. Protein,as well as, antibodies directed against the protein may show utility asa tumor marker and/or immunotherapy targets for the above listedtissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:40 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 628 of SEQID NO:40, b is an integer of 15 to 642, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:40, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 31

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: ARDRTHCLL (SEQ ID NO:203).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

This gene is expressed primarily in epididymus, and to a lesser extent,in colon tissue.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, infertility; spermdevelopmental/survival disorders. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe reproductive system, expression of this gene at significantly higheror lower levels may be routinely detected in certain tissues or celltypes or cell types (e.g., reproductive, cancerous and wounded tissues)or bodily fluids (e.g., lymph, seminal fluid, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in epididymus suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosisand/or treatment of male infertility. Specific expression of this geneproduct within the epididymus suggests that it plays key roles in thedevelopment and/or survival of sperm. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:41 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 428 of SEQID NO:41, b is an integer of 15 to 442, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:41, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 32

This gene is expressed primarily in T-cells, osteoblasts, pancreas,colon and to a lesser extent, in healing groin wound library and manyother tissues.

FIGS. 1A-C show the nucleotide (SEQ ID NO:42) and deduced amino acidsequence (SEQ ID NO: 98) corresponding to this gene.

FIG. 2 shows an analysis of the amino acid sequence (SEQ ID NO: 98).

The data presented in FIG. 2 are also represented in tabular form inTable 6.

Also preferred are polypeptides comprising, or alternatively consistingof, the mature form of the polypeptide having the amino acid sequence ofSEQ ID NO:98, the mature form of the polypeptide encoded by the cDNAcontained in ATCC Deposit No. 209277, and/or the mature polypeptidewhich is predicted to consist of residues 19-300 of the foregoingsequence (SEQ ID NO:98), and biologically active fragments of thesepolypeptides (e.g., fragments that induce T-cell proliferation).Polynucleotides encoding these polypeptides are also encompassed by theinvention. Moreover, fragments and variants of these polypeptides (suchas, for example, fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by nucleic acids which hybridize,under stringent conditions, to the polynucleotide encoding thesepolypeptides, or the complement thereof are encompassed by theinvention. Polynucleotides encoding these polypeptides are alsoencompassed by the inventions.

The present invention is further directed to fragments of thepolynucleotide sequences described herein. By a fragment of, forexample, the polynucleotide sequence of a deposited cDNA or thenucleotide sequence shown in SEQ ID NO:42, is intended polynucleotidefragments at least about 15 nt, and more preferably at least about 20nt, at least about 25 nt, still more preferably at least about 30 nt, atleast about 35 nt, and even more preferably, at least about 40 nt inlength, at least about 45 nt in length, at least about 50 nt in length,at least about 60 nt in length, at least about 70 nt in length, at leastabout 80 nt in length, at least about 90 nt in length, at least about100 nt in length, at least about 125 nt in length, at least about 150 ntin length, at least about 175 nt in length, which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 200-1500 nt in length are also useful according to the presentinvention, as are fragments corresponding to most, if not all, of thenucleotide sequence of a deposited cDNA or as shown in SEQ ID NO:42. Bya fragment at least 20 nt in length, for example, is intended fragmentswhich include 20 or more contiguous bases from the nucleotide sequenceof a deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:42.In this context “about” includes the particularly recited size, an sizeslarger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini. Representative examples of polynucleotidefragments of the invention include, for example, fragments thatcomprise, or alternatively, consist of, a sequence from about nucleotide1 to about 50, from about 51 to about 100, from about 101 to about 150,from about 151 to about 200, from about 201 to about 250, from about 251to about 300, from about 301 to about 350, from about 351 to about 400,from about 401 to about 450, from about 451 to about 500, and from about501 to about 550, and from about 551 to about 600, from about 601 toabout 650, from about 651 to about 700, from about 701 to about 750,from about 751 to about 800, from about 801 to about 850, from about 851to about 900, from about 901 to about 950, from about 951 to about 1000,from about 1001 to about 1050, from about 1051 to about 1100, from about1101 to about 1150 from about 1151 to about 1200, from about 1201 toabout 1250, from about 1251 to about 1300, from about 1301 to about1350, from about 1351 to about 1400, from about 1401 to about 1450, andfrom about 1451 to about 1500, from about 1501 to about 1550, from about1551 to about 1600 , from about 1 601 to about 1650 , from about 1651 toabout 1734 of SEQ ID NO:42, or the complementary strand thereto, or thecDNA contained in a deposited clone. In this context “about” includesthe particularly recited ranges, and ranges larger or smaller by several(5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.In additional embodiments, the polynucleotides of the invention encodefunctional attributes of the corresponding protein (e.g., the ability toinduce T-cell proliferation).

Preferred polypeptide fragments of the invention comprise, oralternatively consist of, the secreted protein having a continuousseries of deleted residues from the amino or the carboxy terminus, orboth. Particularly, N-terminal deletions of the polypeptide can bedescribed by the general formula m-300 where m is an integer from 2 to294, where m corresponds to the position of the amino acid residueidentified in SEQ ID NO:98. More in particular, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, an amino acid sequence selected from the group: K-2 toQ-300; F-3 to Q-300; L-4 to Q-300; L-5 to Q-300; D-6 to Q-300; I-7 toQ-300; L-8 to Q-300; L-9 to Q-300; L-10 to Q-300; L-11 to Q-300; P-12 toQ-300 ; L-13 to Q-300; L-14 to Q-300; I-15 to Q-300; V-16 to Q-300; C-17to Q-300; S-18 to Q-300; E-20 to Q-300; S-21 to Q-300; F-22 to Q-300;V-23 to Q-300; K-24 to Q-300; L-25 to Q-300; F-26 to Q-300; I-27 toQ-300; P-28 to Q-300; K-29 to Q-300; R-30 to Q-300; R-31 to Q-300; K-32to Q-300; S-33 to Q-300; V-34 to Q-300; T-35 to Q-300; G-36 to Q-300;E-37 to Q-300; I-38 to Q-300; V-39 to Q-300; L-40 to Q-300; I-41 toQ-300; T-42 to Q-300; G-43 to Q-300; A-44 to Q-300; G-45 to Q-300; H-46to Q-300; G-47 to Q-300; I-48 to Q-300; G-49 to Q-300; R-50 to Q-300;L-51 to Q-300; T-52 to Q-300; A-53 to Q-300; Y-54 to Q-300; E-55 toQ-300; F-56 to Q-300; A-57 to Q-300; K-58 to Q-300; L-59 to Q-300; K-60to Q-300; S-61 to Q-300; K-62 to Q-300; L-63 to Q-300; V-64 to Q-300;L-65 to Q-300; W-66 to Q-300; D-67 to Q-300; I-68 to Q-300; N-69 toQ-300; K-70 to Q-300; H-71 to Q-300; G-72 to Q-300; L-73 to Q-300; E-74to Q-300; E-75 to Q-300; T-76 to Q-300; A-77 to Q-300; A-78 to Q-300;K-79 to Q-300; C-80 to Q-300; K-81 to Q-300; G-82 to Q-300; L-83 toQ-300; G-84 to Q-300; A-85 to Q-300; K-86 to Q-300; V-87 to Q-300; H-88to Q-300; T-89 to Q-300; F-90 to Q-300; V-91 to Q-300; V-92 to Q-300;D-93 to Q-300; C-94 to Q-300; S-95 to Q-300; N-96 to Q-300; R-97 toQ-300; E-98 to Q-300; D-99 to Q-300; I-100 to Q-300; Y-101 to Q-300;S-102 to Q-300; S-103 to Q-300; A-104 to Q-300; K-105 to Q-300; K-106 toQ-300; V-107 to Q-300; K-108 to Q-300; A-109 to Q-300; E-110 to Q-300;I-111 to Q-300; G-112 to Q-300; D-113 to Q-300; V-114 to Q-300; S-115 toQ-300; I-116 to Q-300; L-117 to Q-300; V-118 to Q-300; N-119 to Q-300;N-120 to Q-300; A-121 to Q-300; G-122 to Q-300; V-123 to Q-300; V-124 toQ-300; Y-125 to Q-300; T-126 to Q-300; S-127 to Q-300; D-128 to Q-300;L-129 to Q-300; F-130 to Q-300; A-131 to Q-300; T-132 to Q-300; Q-133 toQ-300; D-134 to Q-300; P-135 to Q-300; Q-136 to Q-300; I-137 to Q-300;E-138 to Q-300; K-139 to Q-300; T-140 to Q-300; F-141 to Q-300; E-142 toQ-300; V-143 to Q-300; N-144 to Q-300; V-145 to Q-300; L-146 to Q-300;A-147 to Q-300; H-148 to Q-300; F-149 to Q-300; W-150 to Q-300; T-151 toQ-300; T-152 to Q-300; K-153 to Q-300; A-154 to Q-300; F-155 to Q-300;L-156 to Q-300; P-157 to Q-300; A-158 to Q-300; M-159 to Q-300; T-160 toQ-300; K-161 to Q-300; N-162 to Q-300; N-163 to Q-300; H-164 to Q-300;G-165 to Q-300; H-166 to Q-300; I-167 to Q-300; V-168 to Q-300; T-169 toQ-300; V-170 to Q-300; A-171 to Q-300; S-172 to Q-300; A-173 to Q-300;A-174 to Q-300; G-175 to Q-300; H-176 to Q-300; V-177 to Q-300; S-178 toQ-300; V-179 to Q-300; P-180 to Q-300; F-181 to Q-300; L-182 to Q-300;L-183 to Q-300; A-184 to Q-300; Y-185 to Q-300; C-186 to Q-300; S-187 toQ-300; S-188 to Q-300; K-189 to Q-300; F-190 to Q-300; A-191 to Q-300;A-192 to Q-300; V-193 to Q-300; G-194 to Q-300; F-195 to Q-300; H-196 toQ-300; K-197 to Q-300; T-198 to Q-300; L-199 to Q-300; T-200 to Q-300;D-201 to Q-300; E-202 to Q-300; L-203 to Q-300; A-204 to Q-300; A-205 toQ-300; L-206 to Q-300; Q-207 to Q-300; I-208 to Q-300; T-209 to Q-300;G-210 to Q-300; V-211 to Q-300; K-212 to Q-300; T-213 to Q-300; T-214 toQ-300; C-215 to Q-300; L-216 to Q-300; C-217 to Q-300; P-218 to Q-300;N-219 to Q-300; F-220 to Q-300; V-221 to Q-300; N-222 to Q-300; T-223 toQ-300; G-224 to Q-300; F-225 to Q-300; I-226 to Q-300; K-227 to Q-300;N-228 to Q-300; P-229 to Q-300; S-230 to Q-300; T-231 to Q-300; S-232 toQ-300; L-233 to Q-300; G-234 to Q-300; P-235 to Q-300; T-236 to Q-300;L-237 to Q-300; E-238 to Q-300; P-239 to Q-300; E-240 to Q-300; E-241 toQ-300; V-242 to Q-300; V-243 to Q-300; N-244 to Q-300; R-245 to Q-300;L-246 to Q-300; M-247 to Q-300; H-248 to Q-300; G-249 to Q-300; I-250 toQ-300; L-251 to Q-300; T-252 to Q-300; E-253 to Q-300; Q-254 to Q-300;K-255 to Q-300; M-256 to Q-300; I-257 to Q-300; F-258 to Q-300; I-259 toQ-300; P-260 to Q-300; S-261 to Q-300; S-262 to Q-300; I-263 to Q-300;A-264 to Q-300; F-265 to Q-300; L-266 to Q-300; T-267 to Q-300; T-268 toQ-300; L-269 to Q-300; E-270 to Q-300; R-271 to Q-300; I-272 to Q-300;L-273 to Q-300; P-274 to Q-300; E-275 to Q-300; R-276 to Q-300; F-277 toQ-300; L-278 to Q-300; A-279 to Q-300; V-280 to Q-300; L-281 to Q-300;K-282 to Q-300; R-283 to Q-300; K-284 to Q-300; I-285 to Q-300; S-286 toQ-300; V-287 to Q-300; K-288 to Q-300; F-289 to Q-300; D-290 to Q-300;A-291 to Q-300; V-292 to Q-300; I-293 to Q-300; G-294 to Q-300; andY-295 to Q-300 ; of SEQ ID NO:98. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention. Moreover,fragments and variants of these polypeptides (such as, for example,fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to these polypeptides and polypeptidesencoded by nucleic acids which hybridize, under stringent conditions, tothe polynucleotide encoding these polypeptides, or the complementthereof are encompassed by the invention. Polynucleotides encoding thesepolypeptides are also encompassed by the inventions. Antibodies thatbind polypeptides of the invention are also encompassed by theinvention. In preferred embodiments, the invention is directed toantibodies that bind and inhibit activity (e.g., T-cell proliferation).

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification, of loss of oneor more biological functions of the protein (e.g., ability to stimulateT-cell proliferation), other functional activities (e.g., biologicalactivities, ability to multimerize, ability to bind ligand, ability togenerate antibodies, ability to bind antibodies) may still be retained.For example the ability of the shortened polypeptide to induce and/orbind to antibodies which recognize the complete or mature forms of thepolypeptide generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theC-terminus. Whether a particular polypeptide lacking C-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a polypeptide with a largenumber of deleted C-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the polypeptide shown in FIGS. 1A-C (SEQ ID NO: 98), asdescribed by the general formula 1−n, where n is an integer from 6 to299, where n corresponds to the position of the amino acid residueidentified in SEQ ID NO:98. More in particular, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, an amino acid sequence selected from the group: M-1 toA-299; M-1 to K-298; M-1 to M-297; M-1 to K-296; M-1 to Y-295; M-1 toG-294; M-1 to I-293; M-1 to V-292; M-1 to A-291; M-1 to D-290; M-1 toF-289; M-1 to K-288; M-1 to V-287; M-1 to S-286; M-1 to 1-285; M-1 toK-284; M-1 to R-283; M-1 to K-282; M-1 to L-281; M-1 to V-280; M-1 toA-279; M-1 to L-278; M-1 to F-277; M-1 to R-276; M-1 to E-275; M-1 toP-274; M-1 to L-273; M-1 to I-272; M-1 to R-271; M-1 to E-270; M-1 toL-269; M-1 to T-268; M-1 to T-267; M-1 to L-266; M-1 to F-265; M-1 toA-264; M-1 to I-263; M-1 to S-262; M-1 to S-261; M-1 to P-260; M-1 to1-259; M-1 to F-258; M-1 to I-257; M-1 to M-256; M-1 to K-255; M-1Q-254; M-1 to E-253; M-1 to T-252; M-1 to L-251; M-1 to I-250; M-1 toG-249; M-1 H-248; M-1 to M-247; M-1 to L-246; M-1 to R-245; M-1 toN-244; M-1 to V-243; M-1 to V-242; M-1 to E-241; M-1 to E-240; M-1 toP-239; M-1 to E-238; M-1 to L237; M-1 to T-236; M-1 to P-235; M-1 toG-234; M-1 to L-233; M-1 to S-232; M-1 to T-231; M-1 to S-230; M-1 toP-229; M-1 to N-228; M-1 to K-227; M-1 to I-226; M-1 to F-225; M-1 toG-224; M-1 to T-223; M-1 to N-222; M-1 to V-221; M-1 to F-220; M-1 toN-219; M-1 to P-218; M-1 to C-217; M-1 to L-216; M-1 to C-215; M-1 toT-214; M-1 to T-213; M-1 to K-212; M-1 to V-211; M-1 to G-210; M-1 toT-209; M-1 to 1-208; M-1 to Q-207; M-1 to L-206; M-1 to A-205; M-1 toA-204; M-1 to L-203; M-1 to E-202; M-1 to D-201; M-1 to T-200; M-1 toL-199; M-1 to T-198; M-1 to K-197; M-1 to H-196; M-1 to F-195; M-1 toG-194; M-1 to V-193; M-1 to A-192; M-1 to A-191; M-1 to F-190; M-1 toK-189; M-1 to S-188; M-1 to S-187; M-1 to C-186; M-1 to Y-185; M-1 toA-184; M-1 to L-183; M-1 to L-182; M-1 to F-181; M-1 to P-180; M-1 toV-179; M-1 to S-178; M-1 to V-177; M-1 to H-176; M-1 to G-175; M-1 toA-174; M-1 to A-173; M-1 to S-172; M-1 to A-171; M-1 to V-170; M-1 toT-169; M-1 to V-168; M-1 to 1-167; M-1 to H-166; M-1 to G-165; M-1 toH-164; M-1 to N-163; M-1 to N-162; M-1 to K-161; M-1 to T-160; M-1 toM-159; M-1 to A-158; M-1 to P-157; M-1 to L-156; M-1 to F-155; M-1 toA-154; M-1 to K-153; M-1 to T-152; M-1 to T-151; M-1 to W-150; M-1 toF-149; M-1 to H-148; M-1 to A-147; M-1 to L-146; M-1 to V-145; M-1 toN-144; M-1 to V-143; M-I to E-142; M-1 to F-141; M-1 to T-140; M-1 toK-139; M-1 to E-138; M-1 to 1-137; M-1 to Q-136; M-1 to P-135; M-1 toD-134; M-1 to Q-133; M-1 to T-132; M-1 to A-131; M-1 to F-130; M-1 toL-129; M-1 to D-128; M-1 to S-127; M-1 to T-126; M-1 to Y-125; M-1 toV-124; M-1 to V-123; M-1 to G-122; M-1 to A-121; M-1 to N-120; M-1 toN-119; M-1 to V-118; M-1 to L-117; M-1 to I-116; M-1 to S-115; M-1 toV-114; M-1 to D-113; M-1 to G-112; M-1 to I-111; M-1 to E-110; M-1 toA-109; M-1 to K-108; M-1 to V-107; M-1 to K-106; M-1 to K-105; M-1 toA-104; M-1 to S-103; M-1 to S-102; M-1 to Y-101; M-1 to I-100; M-1 toD-99; M-1 to E-98; M-1 to R-97; M-1 to N-96; M-1 to S-95; M-1 to C-94;M-1 to D-93; M-1 to V-92; M-1 to V-91; M-1 to F-90; M-1 to T-89; M-1 toH-88; M-1 to V-87; M-1 to K-86; M-1 to A-85; M-1 to G-84; M-1 to L-83;M-1 to G-82; M-1 to K-81; M-1 to C-80; M-1 to K-79; M-1 to A-78; M-1 toA-77; M-1 to T-76; M-1 to E-75; M-1 to E-74; M-1 to L-73; M-1 to G-72;M-1 to H-71; M-1 to K-70; M-1 to N-69; M-1 to 1-68; M-1 to D-67; M-1 toW-66; M-1 to L-65; M-1 to V-64; M-1 to L-63; M-1 to K-62; M-1 to S-61;M-1 to K-60; M-1 to L-59; M-1 to K-58; M-1 to A-57; M-1 to F-56; M-1 toE-55; M-1 to Y-54; M-1 to A-53; M-1 to T-52; M-1 to L-51; M-1 to R-50;M-1 to G-49; M-1 to 1-48; M-1 to G-47; M-1 to H-46; M-1 to G-45; M-1 toA-44; M-1 to G-43; M-1 to T-42; M-1 to 1-41; M-1 to L-40; M-1 to V-39;M-1 to I-38; M-1 to E-37; M-1 to G-36; M-1 to T-35; M-1 to V-34; M-1 toS-33; M-1 to K-32; M-1 to R-31; M-1 to R-30; M-1 to K-29; M-1 to P-28;M-1 to I-27; M-1 to F-26; M-1 to L-25; M-1 to K-24; M-1 to V-23; M-1 toF-22; M-1 to S-21; M-1 to E-20; M-1 to L-19; M-1 to S-18; M-1 to C-17;M-1 to V-16; M-1 to I-15; M-1 to L-14; M-1 to L-13; M-1 to P-12; M-1 toL-11; M-1 to L-10; M-1 to L-9; M-1 to L-8; M-1 to I-7; and M-1 to D-6 ofSEQ ID NO: 98. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention. Moreover, fragments and variants of thesepolypeptides (such as, for example, fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to these polypeptides and polypeptides encoded by nucleicacids which hybridize, under stringent conditions, to the polynucleotideencoding these polypeptides, or the complement thereof are encompassedby the invention. Polynucleotides encoding these polypeptides are alsoencompassed by the inventions. Antibodies that bind polypeptides of theinvention are also encompassed by the invention. In preferredembodiments, the invention is directed to antibodies that bind andinhibit activity (e.g., T-cell proliferation).

In addition, any of the above listed N- or C-terminal deletions can becombined to produce a N- and C-terminal deleted polypeptide. Theinvention also provides polypeptides comprising, or alternativelyconsisting of, one or more amino acids deleted from both the amino andthe carboxyl termini, which may be described generally as havingresidues m−n of SEQ ID NO:98, where n and m are integers as describedabove. Polynucleotides encoding these polypeptides are also encompassedby the invention.

The present invention is also directed to proteins containingpolypeptides at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identical to a polypeptide sequence set forth herein as m−n. Inpreferred embodiments, the application is directed to proteinscontaining polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identical to polypeptides having the amino acid sequence of thespecific N- and C-terminal deletions recited herein. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

Also included are polynucleotide sequences encoding a polypeptideconsisting of a portion of the complete amino acid sequence encoded by acDNA clone contained in ATCC Deposit No. 209277, where this portionexcludes any integer of amino acid residues from 1 to about 294 aminoacids from the amino terminus of the complete amino acid sequenceencoded by a cDNA clone contained in ATCC Deposit No. 209277, or anyinteger of amino acid residues from 6 to about 300 amino acids from thecarboxy terminus, or any combination of the above amino terminal andcarboxy terminal deletions, of the complete amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No. 209277. Polypeptidesencoded by these polynucleotides also are encompassed by the invention.

Additional preferred polypeptide fragments of the invention comprise, oralternatively consist of, an amino acid sequence selected from thegroup: M-1 to I-15; K-2 to V-16; F-3 to C-17; L-4 to S-18; L-5 to L-19;D-6 to E-20; I-7 to S-21; L-8 to F-22; L-9 to V-23; L-10 to K-24; L-11to L-25; P-12 to F-26; L-13 to I-27; L-14 to P-28; I-15 to K-29; V-16 toR-30; C-17 to R-31; S-18 to K-32; L-19 to S-33; E-20 to V-34; S-21 toT-35; F-22 to G-36; V-23 to E-37; K-24 to I-38; L-25 to V-39; F-26 toL-40; I-27 to I-41; P-28 to T-42; K-29 to G-43; R-30 to A-44; R-31 toG-45; K-32 to H-46; S-33 to G-47; V-34 to I-48; T-35 to G-49; G-36 toR-50; E-37 to L-51; I-38 to T-52; V-39 to A-53; L-40 to Y-54; I-41 toE-55; T-42 to F-56; G-43 to A-57; A-44 to K-58; G-45 to L-59; H-46 toK-60; G-47 to S-61; I-48 to K-62; G-49 to L-63; R-50 to V-64; L-51 toL-65; T-52 to W-66; A-53 to D-67; Y-54 to I-68; E-55 to N-69; F-56 toK-70; A-57 to H-71; K-58 to G-72; L-59 to L-73; K-60 to E-74; S-61 toE-75; K-62 to T-76; L-63 to A-77; V-64 to A-78; L-65 to K-79; W-66 toC-80; D-67 to K-81; I-68 to G-82; N-69 to L-83; K-70 to G-84; H-71 toA-85; G-72 to K-86; L-73 to V-87; E-74 to H-88; E-75 to T-89; T-76 toF-90; A-77 to V-91; A-78 to V-92; K-79 to D-93; C-80 to C-94; K-81 toS-95; G-82 to N-96; L-83 to R-97; G-84 to E-98; A-85 to D-99; K-86 toI-100; V-87 to Y-101; H-88 to S-102; T-89 to S-103; F-90 to A-104; V-91to K-105; V-92 to K-106; D-93 to V-107; C-94 to K-108; S-95 to A-109;N-96 to E-110; R-97 to I-111; E-98 to G-112; D-99 to D-113; I-100 toV-114; Y-101 to S-115; S-102 to I-116; S-103 to L-117; A-104 to V-118;K-105 to N-119; K-106 to N-120; V-107 to A-121; K-108 to G-122; A-109 toV-123; E-110 to V-124; I-111 to Y-125; G-112 to T-126; D-113 to S-127;V-114 to D-128; S-115 to L-129; I-116 to F-130; L-117 to A-131; V-118 toT-132; N-119 to Q-133; N-120 to D-134; A-121 to P-135; G-122 to Q-136;V-123 to I-137; V-124 to E-138; Y-125 to K-139; T-126 to T-140; S-127 toF-141; D-128 to E-142; L-129 to V-143; F-130 to N-144; A-131 to V-145;T-132 to L-146; Q-133 to A-147; D-134 to H-148; P-135 to F-149; Q-136 toW-150; I-137 to T-151; E-138 to T-152; K-139 to K-153; T-140 to A-154;F-141 to F-155; E-142 to L-156; V-143 to P-157; N-144 to A-158; V-145 toM-159; L-146 to T-160; A-147 to K-161; H-148 to N-162; F-149 to N-163;W-150 to H-164; T-151 to G-165; T-152 to H-166; K-153 to I-167; A-154 toV-168; F-155 to T-169; L-156 to V-170; P-157 to A-171; A-158 to S-172;M-159 to A-173; T-160 to A-174; K-161 to G-175; N-162 to H-176; N-163 toV-177; H-164 to S-178; G-165 to V-179; H-166 to P-180; I-167 to F-181;V-168 to L-182; T-169 to L-183; V-170 to A-184; A-171 to Y-185; S-172 toC-186; A-173 to S-187; A-174 to S-188; G-175 to K-189; H-176 to F-190;V-177 to A-191; S-178 to A-192; V-179 to V-193; P-180 to G-194; F-181 toF-195; L-182 to H-196; L-183 to K-197; A-184 to T-198; Y-185 to L-199;C-186 to T-200; S-187 to D-201; S-188 to E-202; K-189 to L-203; F-190 toA-204; A-191 to A-205; A-192 to L-206; V-193 to Q-207; G-194 to I-208;F-195 to T-209; H-196 to G-210; K-197 to V-211; T-198 to K-212; L-199 toT-213; T-200 to T-214; D-201 to C-215; E-202 to L-216; L-203 to C-217;A-204 to P-218; A-205 to N-219; L-206 to F-220; Q-207 to V-221; I-208 toN-222; T-209 to T-223; G-210 to G-224; V-211 to F-225; K-212 to I-226;T-213 to K-227; T-214 to N-228; C-215 to P-229; L-216 to S-230; C-217 toT-231; P-218 to S-232; N-219 to L-233; F-220 to G-234; V-221 to P-235;N-222 to T-236; T-223 to L-237; G-224 to E-238; F-225 to P-239; I-226 toE-240; K-227 to E-241; N-228 to V-242; P-229 to V-243; S-230 to N-244;T-231 to R-245; S-232 to L-246; L-233 to M-247; G-234 to H-248; P-235 toG-249; T-236 to I-250; L-237 to L-251; E-238 to T-252; P-239 to E-253;E-240 to Q-254; E-241 to K-255; V-242 to M-256; V-243 to I-257; N-244 toF-258; R-245 to I-259; L-246 to P-260; M-247 to S-261; H-248 to S-262;G-249 to I-263; I-250 to A-264; L-251 to F-265; T-252 to L-266; E-253 toT-267; Q-254 to T-268; K-255 to L-269; M-256 to E-270; I-257 to R-271;F-258 to I-272; 1-259 to L-273; P-260 to P-274; S-261 to E-275; S-262 toR-276; I-263 to F-277; A-264 to L-278; F-265 to A-279; L-266 to V-280;T-267 to L-281; T-268 to K-282; L-269 to R-283; E-270 to K-284; R-271 toI-285; I-272 to S-286; L-273 to V-287; P-274 to K-288; E-275 to F-289;R-276 to D-290; F-277 to A-291; L-278 to V-292; A-279 to I-293; V-280 toG-294; L-281 to Y-295; K-282 to K-296; R-283 to M-297; K-284 to K-298;I-285 to A-299; and S-286 to Q-300 of SEQ ID NO: 98. Polypeptidesencoded by these polynucleotides are also encompassed by the invention.Moreover, fragments and variants of these polypeptides (such as, forexample, fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by nucleic acids which hybridize, under stringentconditions, to the polynucleotide encoding these polypeptides, or thecomplement thereof are encompassed by the invention. Polynucleotidesencoding these polypeptides are also encompassed by the inventions.Antibodies that bind polypeptides of the invention are also encompassedby the invention. In preferred embodiments, the invention is directed toantibodies that bind and inhibit activity (e.g., T-cell proliferation).

Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, or all four of theimmunogenic epitopes shown in SEQ ID NO: 98 as residues: Lys-29 toVal-34, Cys-94 to Asp-99, Ser-102 to Val-107, and Gln-133 to Lys-139.Polynucleotides encoding these polypeptides are also encompassed by theinvention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(such as, for example, fragments as described herein, polypeptides atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by nucleic acids which hybridize,under stringent conditions, to the polynucleotide encoding thesepolypeptides, or the complement thereof are encompassed by theinvention. Polynucleotides encoding these polypeptides are alsoencompassed by the inventions.

In additional nonexclusive embodiments, polypeptides of the invention,comprise or alternatively consist of, the following amino acid sequence:THQTLAATKG (SEQ ID NO:204). Polynucleotides encoding these polypeptidesare also encompassed by the invention, as are antibodies that bind thesepolypeptides.

FIG. 2 shows an analysis of the amino acid sequence (SEQ ID NO: 98).Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown, and all were generated using the default settingsof the recited computer algorithyms. In the “Antigenic Index orJameson-Wolf” graph, the positive peaks indicate locations of the highlyantigenic regions of the protein, i.e., regions from whichepitope-bearing peptides of the invention can be obtained. Polypeptidescomprising, or alternatively consisting of, domains defined by thesegraphs are contemplated by the present invention, as are polynucleotidesencoding these polypeptides.

The data presented in FIG. 2 are also represented in tabular form inTable 6. The columns are labeled with the headings “Res”, “Position”,and Roman Numerals I-XIV. The column headings refer to the followingfeatures of the amino acid sequence presented in FIG. 2, and Table 6:“Res”: amino acid residue of SEQ ID NO: 98 and FIGS. 1A-C; “Position”:position of the corresponding residue within SEQ ID NO: 98 and FIGS.1A-C; I: Alpha, Regions—Garnier-Robson; II: Alpha, Regions—Chou-Fasman;III: Beta, Regions—Garnier-Robson; IV: Beta, Regions—Chou-Fasman; V:Turn, Regions—Garnier-Robson; VI: Turn, Regions—Chou-Fasman; VII: Coil,Regions—Garnier-Robson; VIII: Hydrophilicity Plot—Kyte-Doolittle; IX:Hydrophobicity Plot—Hopp-Woods; X: Alpha, Amphipathic Regions—Eisenberg;XI: Beta, Amphipathic Regions—Eisenberg; XII: FlexibleRegions—Karplus-Schulz; XIII: Antigenic Index—Jameson-Wolf; and XIV:Surface Probability Plot—Emini.

Preferred embodiments of the invention in this regard include fragmentsthat comprise, or alternatively consisting of, one or more of thefollowing regions: alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions. The data representing the structural or functionalattributes of the protein set forth in FIG. 2 and/or Table 6, asdescribed above, was generated using the various modules and algorithmsof the DNA*STAR set on default parameters. In a preferred embodiment,the data presented in columns VIII, IX, XIII, and XIV of Table 6 can beused to determine regions of the protein which exhibit a high degree ofpotential for antigenicity. Regions of high antigenicity are determinedfrom the data presented in columns VIII, IX, XIII, and/or XIV bychoosing values which represent regions of the polypeptide which arelikely to be exposed on the surface of the polypeptide in an environmentin which antigen recognition may occur in the process of initiation ofan immune response.

Certain preferred regions in these regards are set out in FIG. 2, butmay, as shown in Table 6, be represented or identified by using tabularrepresentations of the data presented in FIG. 2. The DNA*STAR computeralgorithm used to generate FIG. 2 (set on the original defaultparameters) was used to present the data in FIG. 2 in a tabular format(See Table 6). The tabular format of the data in FIG. 2 is used toeasily determine specific boundaries of a preferred region.

The gene encoding the disclosed cDNA is thought to reside on chromosome4. Accordingly, polynucleotides related to this invention are useful asa marker in linkage analysis for chromosome 4.

As described herein or otherwise known in the art, the polynucleotidesof the invention have uses that include, but are not limited to, servingas probes or primers in chromosome identification, chromosome mapping,and linkage analysis.

Additionally, polypeptides of the invention have been found induceT-cell proliferation. Representative examples of assays used todemonstrate T-cell proliferative activity are described in Example 33herein.

Accordingly, compositions of the invention (including polynucleotides,polypeptides and antibodies of the invention, and fragments and variantsthereof) may be used in the diagnosis, detection and/or treatment ofdiseases and/or immune disorders associated with aberrant T-cellproliferation and/or activation. In preferred embodiments, compositionsof the invention (including polynucleotides, polypeptides and antibodiesof the invention, and fragments and variants thereof) may be used in thediagnosis, detection and/or treatment of diseases and/or disordersrelating to the immune system in general, and T-cell proliferationand/or activation specifically (e.g., cytokine production, inflammation,T cell proliferation and T cell proliferative disorders, and/or asdescribed under “Immune Activity”, “Hyperproliferative Disorders” and“Diseases at the Cellular Level” below). Thus, polynucleotides,translation products and antibodies of the invention are useful in thediagnosis, detection and/or treatment of diseases and/or disordersassociated with activities that include, but are not limited to, T cellactivation, cytokine production, T cell proliferation, T cellproliferative disorders, T cell related immunodeficiencies (e.g.,autoimmune disorders), inflammation, graft rejection and immune systemdisorders.

Polynucleotides and polypeptides of the invention are also useful asreagents for differential identification of the tissue(s) or celltype(s) present in a biological sample and for diagnosis of diseases andconditions which include, but are not limited to, immunological andwound healing diseases and/or disorders. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types or cell types (e.g., immune, metabolic,cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution in immune cells (e.g., T-cells) and the factthat polypeptides of this invention have been found induce T-cellproliferation indicates that polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention would be usefulfor the diagnosis, treatment, and/or prevention of a variety of immunesystem disorders. In particular, polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present invention maybe useful in treating, preventing, and/or diagnosing diseases,disorders, and/or conditions of the immune system, by, for example,activating or inhibiting the proliferation, activation, differentiation,or mobilization (chemotaxis) of immune cells. Representative uses aredescribed in the “Immune Activity” and “Infectious Disease” sectionsbelow, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhereherein. Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, and/or diagnosing diseases, disorders, and/or conditions ofhematopoietic cells. For example, polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present inventioncould be used to increase differentiation and proliferation ofhematopoietic cells, including the pluripotent stem cells, in an effortto treat or prevent those diseases, disorders, and/or conditionsassociated with a decrease in certain (or many) types hematopoieticcells. Examples of immunologic deficiency syndromes include, but are notlimited to: blood protein diseases, disorders, and/or conditions (e.g.,agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, commonvariable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLVinfection, leukocyte adhesion deficiency syndrome, lymphopenia,phagocyte bactericidal dysfunction, severe combined immunodeficiency(SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, orhemoglobinuria.

The expression in T-cells and the fact that polypeptides of thisinvention have been found to induce T-cell proliferation indicates arole in regulating the proliferation; survival; differentiation; and/oractivation of hematopoietic cell lineages, including blood stem cells.Involvement in the regulation of cytokine production, antigenpresentation, or other processes suggests a usefulness for treatment ofcancer (e.g. by boosting immune responses). Expression in cells oflymphoid origin, indicates the natural gene product would be involved inimmune functions. Therefore it would also be useful as an agent forimmunological disorders including arthritis, asthma, immunodeficiencydiseases such as AIDS, leukemia, rheumatoid arthritis, granulomatousdisease, inflammatory bowel disease, sepsis, acne, neutropenia,neutrophilia, psoriasis, hypersensitivities, such as T-cell mediatedcytotoxicity; immune reactions to transplanted organs and tissues, suchas host-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.Moreover, the protein may represent a secreted factor that influencesthe differentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product isthought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types.

The tissue distribution in T-cells suggests that polynucleotides andpolypeptides corresponding to this gene are useful for modulating theimmune response during wound repair. The translation product of thisgene may function as a stimulator for the growth of bone, cartilage,tendons, ligaments and/or nerves, which would be useful for thetreatment of wounds. Moreover, the protein may be useful in detection,treating, and/or preventing metabolic conditions, particularlyconditions related to aberrant fatty-acid metabolism. The protein isuseful in modulating the immune response to aberrant polypeptides, asmay exist in proliferating and cancerous cells and tissues.

In specific non-exclusive embodiments, inflammatory conditions may alsobe treated, diagnosed, and/or prevented with polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention. Such inflammatory conditions include, but are not limited to,for example, respiratory disorders (such as, e.g., asthma and allergy);gastrointestinal disorders (such as, e.g., inflammatory bowel disease);cancers (such as, e.g., gastric, ovarian, lung, bladder, liver, andbreast); CNS disorders (such as, e.g., multiple sclerosis, blood-brainbarrier permeability, ischemic brain injury and/or stroke, traumaticbrain injury, neurodegenerative disorders (such as, e.g., Parkinson'sdisease and Alzheimer's disease), AIDS-related dementia, and priondisease); cardiovascular disorders (such as, e.g., atherosclerosis,myocarditis, cardiovascular disease, and cardiopulmonary bypasscomplications); as well as many additional diseases, conditions, anddisorders that are characterized by inflammation (such as, e.g., chronichepatitis (B and C), rheumatoid arthritis, gout, trauma, septic shock,pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusioninjury, Grave's disease, systemic lupus erythematosis, diabetes mellitus(i.e., type 1 diabetes), and allogenic transplant rejection).

More specifically, polypeptides, antibodies, or polynucleotides of theinvention, and/or agonists or antagonists thereof, are useful to treat,diagnose, and/or prevent transplantation rejections, graft-versus-hostdisease, autoimmune and inflammatory diseases (e.g., immunecomplex-induced vasculitis, glomerulonephritis, hemolytic anemia,myasthenia gravis, type II collagen-induced arthritis, experimentalallergic and hyperacute xenograft rejection, rheumatoid arthritis, andsystemic lupus erythematosus (SLE). Organ rejection occurs by hostimmune cell destruction of the transplanted tissue through an immuneresponse. Similarly, an immune response is also involved in GVHD, but,in this case, the foreign transplanted immune cells destroy the hosttissues. Polypeptides, antibodies, or polynucleotides of the invention,and/or agonists or antagonists thereof, that inhibit an immune response,particularly the activation, proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventing organrejection or GVHD.

Similarly, polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may also be used to modulate and/ordiagnose inflammation. For example, since polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagonists of theinvention may inhibit the activation, proliferation and/ordifferentiation of cells involved in an inflammatory response, thesemolecules can be used to treat, diagnose, or prognose, inflammatoryconditions, both chronic and acute conditions, including, but notlimited to, inflammation associated with infection (e.g., septic shock,sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, and resulting from over production of cytokines (e.g., TNF orIL-1).

Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues. Furthermore, the protein may also be used to determinebiological activity, raise antibodies, as tissue markers, to isolatecognate ligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:42 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 1720 of SEQID NO:42, b is an integer of 15 to 1734, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:42, and whereb is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 33

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: MPRPSPLSSPGSPVTSQLCSPMPSLNPALPWGLLLALPGLSLHTPFQTLTAASPHQPSGDSAAHLSAHSFLLDSH (SEQ ID NO: 205); VPCGTACSVGAAA(SEQ ID NO:206); and/or TSRSMF TSRPRTPWTSCLQIAP LALLQSLGIWQHSIGA (SEQ IDNO:207). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

This gene is expressed primarily in neutrophils.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, immune-related diseases and/ordisorders. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types orcell types (e.g., immune, cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in neutrophils suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosis,treatment, and/or prevention of immune related diseases. Moreover, theexpression of this gene product in neutrophils suggests a role inregulating the proliferation; survival; differentiation; and/oractivation of potentially all hematopoietic cell lineages, includingblood stem cells. This gene product may be involved in the regulation ofcytokine production, antigen presentation, or other processes that mayalso suggest a usefulness in the treatment of cancer (e.g., by boostingimmune responses).

Since the gene is expressed in cells of lymphoid origin, the naturalgene or protein, as well as, antibodies directed against the protein mayshow utility as a tumor marker and/or immunotherapy targets for theabove listed tissues. Therefore it may be also used as an agent forimmunological disorders including arthritis, asthma, immune deficiencydiseases such as AIDS, leukemia, rheumatoid arthritis, inflammatorybowel disease, sepsis, acne, and psoriasis. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:43 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 503 of SEQID NO:43, b is an integer of 15 to 517, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:43, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 34

This gene is expressed primarily in 12 week-old human embryos.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, developmental abnormalities;cancer. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theembryo, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types or cell types(e.g., embryonic, cancerous and wounded tissues) or bodily fluids (e.g.,lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in embryonic tissue suggests thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis, detection, and/or treatment of developmentaldisorders. The relatively specific expression of this gene productduring embryogenesis suggests that it may be a key player in theproliferation, maintenance, and/or differentiation of various cell typesduring development. It may also act as a morphogen to control cell andtissue type specification. Expression within embryonic tissue and othercellular sources marked by proliferating cells suggests that thisprotein may play a role in the regulation of cellular division. Becauseof potential roles in proliferation and differentiation, this geneproduct may have applications in the adult for tissue regeneration andthe treatment of cancers.

Similarly, developmental tissues rely on decisions involving celldifferentiation and/or apoptosis in pattern formation. Dysregulation ofapoptosis can result in inappropriate suppression of cell death, asoccurs in the development of some cancers, or in failure to control theextent of cell death, as is believed to occur in acquiredimmunodeficiency and certain neurodegenerative disorders, such as spinalmuscular atrophy (SMA). Therefore, the polynucleotides and polypeptidesof the present invention are useful in treating, detecting, and/orpreventing said disorders and conditions, in addition to other types ofdegenerative conditions. Thus this protein may modulate apoptosis ortissue differentiation and would be useful in the detection, treatment,and/or prevention of degenerative or proliferative conditions anddiseases. The protein is useful in modulating the immune response toaberrant polypeptides, as may exist in proliferating and cancerous cellsand tissues. The protein can also be used to gain new insight into theregulation of cellular growth and proliferation. Protein, as well as,antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:44 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 472 of SEQID NO:44, b is an integer of 15 to 486, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:44, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 35

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: GTAGFSDLLLVNVMCQTRRSITFKNKLQKESRIYP (SEQID NO:208). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

This gene is expressed primarily in CD34 depleted buffy coat (cordblood).

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, blood and immune diseases and/ordisorders. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types orcell types (e.g., immune, cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, amniotic fluid, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in CD34 depleted buffy coat cells suggests thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment of haemopoietic and immune disorders. Expression ofthis gene product in CD34 depleted buffy coat (cord blood) suggests arole in the regulation of the proliferation; survival; differentiation;and/or activation of potentially all hematopoietic cell lineages,including blood stem cells. This gene product may be involved in theregulation of cytokine production, antigen presentation, or otherprocesses that may also suggest a usefulness in the treatment of cancer(e.g., by boosting immune responses).

Since the gene is expressed in cells of lymphoid origin, the naturalgene or protein, as well as, antibodies directed against the protein mayshow utility as a tumor marker and/or immunotherapy targets for theabove listed tissues. Therefore it may be also used as an agent forimmunological disorders including arthritis, asthma, immune deficiencydiseases such as AIDS, leukemia, rheumatoid arthritis, inflammatorybowel disease, sepsis, acne, and psoriasis. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:45 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 812 of SEQID NO:45, b is an integer of 15 to 826, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:45, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 36

This gene is expressed primarily in adipose tissue, and to a lesserextent, in placental tissue.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, metabolic or reproductivedisorders, particularly obesity. Similarly, polypeptides and antibodiesdirected to these polypeptides are useful in providing immunologicalprobes for differential identification of the tissue(s) or cell type(s).For a number of disorders of the above tissues or cells, particularly ofthe metabolic system, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell typesor cell types (e.g., adipose, cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in adipose tissue suggests that polynucleotidesand polypeptides corresponding to this gene are useful for the treatmentof obesity by targeting adipose cells. Furthermore, the protein productof this clone may show utility in ameliorating conditions which occursecondary to aberrant fatty-acid metabolism (e.g., aberrant myelinsheath development), either directly or indirectly. Protein, as well as,antibodies directed against the protein may show utility as atissue-specific marker and/or immunotherapy target for the above listedtissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:46 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 680 of SEQID NO:46, b is an integer of 15 to 694, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:46, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 37

This gene is expressed primarily in adult pulmonary tissue.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, pulmonary disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the pulmonary system, expression ofthis gene at significantly higher or lower levels may be routinelydetected in certain tissues or cell types or cell types (e.g., lung,cancerous and wounded tissues) or bodily fluids (e.g., lymph, pulmonarysurfactant or sputum, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in pulmonary tissue suggests thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and/or treatment of certain pulmonary disorders, suchas lung cancer, ARDS, and emphysema. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:47 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 842 of SEQID NO:47, b is an integer of 15 to 856, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:47, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 38

This gene is expressed primarily in amygdala tissue of the brain, and toa lesser extent, in infant brain tissues.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, developmental andneurodegenerative diseases of the brain and nervous system. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the central nervous system (CNS),expression of this gene at significantly higher or lower levels may beroutinely detected in certain tissues or cell types or cell types (e.g.,brain, cancerous and wounded tissues) or bodily fluids (e.g., lymph,serum, plasma, urine, synovial fluid and spinal fluid) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution in neural tissues suggests that polynucleotidesand polypeptides corresponding to this gene are useful for the treatmentand/or diagnosis of behavioral or nervous system disorders, such asdepression, schizophrenia, Alzheimer's disease, dementia, paranoia,autism, and addictive behavior. The amygdala processes sensoryinformation and relays this to other areas of the brain including theendocrine and autonomic domains of the hypothalamus and the brain stem.Thus, the translation product of this gene may also be useful for thedetection and/or treatment of neural disorders that impact processesmediated by the amygdala. Protein, as well as, antibodies directedagainst the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:48 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 1629 of SEQID NO:48, b is an integer of 15 to 1643, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:48, and whereb is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 39

This gene is expressed primarily in synovial hypoxia tissue.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, connective tissue disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the connectivetissue system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,skeletal, connective, and cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in synovial hypoxia tissue suggests thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and/or diagnosis of diseases of connective tissue,particularly synovia, including but not limited to inflammation,rheumatoid arthritis, osteoarthritis, and cartilage tears and physicalinjury, as well as osteoporosis, tendonitis, chrondomalacia andinflammation. Furthermore, the translation product of this gene may beuseful in the diagnosis and/or treatment of various autoimmune disorderssuch as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis aswell as dwarfism, spinal deformation, and specific joint abnormalitiesas well as chondrodysplasias (ie. spondyloepiphyseal dysplasiacongenita, familial osteoarthritis, Atelosteogenesis type II,metaphyseal chondrodysplasia type Schmid). Protein, as well as,antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:49 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 695 of SEQID NO:49, b is an integer of 15 to 709, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:49, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 40

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: PFRNSRVRPKGSRDALSWSSCTGPQPGTSATVGSLLCGGVPCIAGHPAASPASCSVPVAPHPAVVTAQVSRCAECPLVMLRGTGVLPPGFE RCLTPTSGVSLPCV (SEQID NO:209). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

This gene is expressed primarily in synovial cells stimulated with IL-1and TNF.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, inflammation of connectivetissue. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly ofconnective tissue, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell typesor cell types (e.g., connective, skeletal, cancerous and woundedtissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in synovial cells suggests that polynucleotidesand polypeptides corresponding to this gene are useful for the treatmentand/or diagnosis of inflammation of connective tissues, particularly thesynovium, in diseases such as rheumatoid arthritis, sepsis, infection ofthe joint, and tissue damage from physical injury. Furthermore, theexpression of this gene product may be useful in the diagnosis ortreatment of various autoimmune disorders such as rheumatoid arthritis,lupus, scleroderma, and dermatomyositis as well as dwarfism, spinaldeformation, and specific joint abnormalities as well aschondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familialosteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasiatype Schmid). Protein, as well as, antibodies directed against theprotein may show utility as a tumor marker and/or immunotherapy targetsfor the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:50 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 527 of SEQID NO:50, b is an integer of 15 to 541, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:50, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 41

When tested against U937 myeloid cell lines as well as Jurkat T-cellcell lines, supernatants removed from cells containing this geneactivated the GAS assay. Therefore, it is likely that this geneactivates myeloid cells, and to a lesser extent, in immune andhematopoietic cells through the Jak-STAT signal transduction pathway.Gamma activating sequence (GAS) is a promoter element found upstream ofmany genes involved in the Jak-STAT pathway. The Jak-STAT pathway is alarge, signal transduction pathway involved in the differentiation andproliferation of cells. Therefore, activation of the Jak-STAT pathway,reflected by the binding of the GAS element, can be used to indicateproteins involved in the proliferation and differentiation of cells.

This gene is expressed primarily in melanocytes, and to a lesser extent,in the synovium, testes, and CD34 cells.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, musculo-skeletal diseases anddisorders, such as skin discoloration or arthritis, as well as malereproductive disorders. Similarly, polypeptides and antibodies directedto these polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of themusculo-skeletal and reproductive systems, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types or cell types (e.g., musculo-skeletal,reproductive, bone, cartilage, cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in synovium suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the treatmentand/or diagnosis of diseases affecting the skeletal system, inparticular osteoporosis, as well as disorders afflicting connectivetissues (e.g., arthritis, trauma, tendonitis, chrondomalacia andinflammation), such as in the diagnosis or treatment of variousautoimmune disorders such as rheumatoid arthritis, lupus, scleroderma,and dermatomyositis as well as dwarfism, spinal deformation, andspecific joint abnormalities as well as chondrodysplasias (ie.spondyloepiphyseal dysplasia congenita, familial osteoarthritis,Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid).

Alternatively, the tissue distribution in testes tissue indicates thatthe protein product of this clone is useful for the treatment anddiagnosis of conditions concerning proper testicular function (e.g.,endocrine function, sperm maturation), as well as cancer. Therefore,this gene product is useful in the treatment of male infertility and/orimpotence. This gene product is also useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. Similarly, the protein is believed to be useful inthe treatment and/or diagnosis of testicular cancer. The testes are alsoa site of active gene expression of transcripts that may be expressed,particularly at low levels, in other tissues of the body. Therefore,this gene product may be expressed in other specific tissues or organswhere it may play related functional roles in other processes, such ashematopoiesis, inflammation, bone formation, and kidney function, toname a few possible target indications. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:51 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 706 of SEQID NO:51, b is an integer of 15 to 720, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:51, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 42

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: MDTYTFLIKICKIFCSFLKCHIQVCGHLLFLIFTSIKWARKQHHCSRCKAIGLSS (SEQ ID NO: 210). Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

This gene is expressed primarily in synovial tissue and neutrophils.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, musculo-skeletal and immunediseases. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune and musculo-skeletal systems, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types or cell types (e.g., immune, skeletal, andcancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution in synovium and neutrophils suggests thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and/or diagnosis of immune diseases, particularlyinflammatory conditions such as arthritis. Expression of this geneproduct in neutrophils suggests a role in the regulation of theproliferation; survival; differentiation; and/or activation ofpotentially all hematopoietic cell lineages, including blood stem cells.This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer (e.g., by boostingimmune responses).

Since the gene is expressed in cells of lymphoid origin, the gene orprotein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues. Therefore it may be also used as an agent forimmunological disorders including arthritis, asthma, immune deficiencydiseases such as AIDS, leukemia, rheumatoid arthritis, inflammatorybowel disease, sepsis, acne, and psoriasis. Furthermore, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types.

In addition, the expression of this gene product in synovium suggests arole in the detection and treatment of disorders and conditionsaffecting the skeletal system, in particular osteoporosis as well asdisorders afflicting connective tissues (e.g., arthritis, trauma,tendonitis, chrondomalacia and inflammation), such as in the diagnosisor treatment of various autoimmune disorders such as rheumatoidarthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism,spinal deformation, and specific joint abnormalities as well aschondrodysplasias (ie. spondyloepiphyseal dysplasia congenita, familialarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia typeSchmid). Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:52 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 965 of SEQID NO:52, b is an integer of 15 to 979, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:52, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 43

This gene is expressed primarily in CD34 depleted buffy coat (cordblood).

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, blood, developmental, and immunedisorders. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types orcell types (e.g., immune, cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in CD34 depleted buffy coat suggests thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the diagnosis and/or treatment of haemopoietic and immune disorders.Furthermore, expression of this gene product in CD34 depleted buffy coat(cord blood) suggests a role in the regulation of the proliferation;survival; differentiation; and/or activation of potentially allhematopoietic cell lineages, including blood stem cells. This geneproduct may be involved in the regulation of cytokine production,antigen presentation, or other processes that may also suggest ausefulness in the treatment of cancer (e.g., by boosting immuneresponses).

Since the gene is expressed in cells of lymphoid origin, the gene orprotein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues. Therefore it may be also used as an agent forimmunological disorders including arthritis, asthma, immune deficiencydiseases such as AIDS, leukemia, rheumatoid arthritis, inflammatorybowel disease, sepsis, acne, and psoriasis. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:53 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 366 of SEQID NO:53, b is an integer of 15 to 380, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:53, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 44

The translation product of this gene shares sequence homology with theconserved NADH-isocitrate dehydrogenase which is thought to be importantin the proliferation of lymphocytes. When tested against Jurkat T-cellcell lines, supernatants removed from cells containing this geneactivated the GAS assay. Therefore, it is likely that this geneactivates T-cells, and to a lesser extent in immune and hematopoieticcells and tissues, through the Jak-STAT signal transduction pathway.Gamma activating sequence (GAS) is a promoter element found upstream ofmany genes involved in the Jak-STAT pathway. The Jak-STAT pathway is alarge, signal transduction pathway involved in the differentiation andproliferation of cells. Therefore, activation of the Jak-STAT pathway,reflected by the binding of the GAS element, can be used to indicateproteins involved in the proliferation and differentiation of cells.

In specific embodiments, polypeptides of the invention comprise, oralternatively consist of, the following amino acid sequences:DPRLAVLLLGVQILVERWRLQWDHYYLCPHRVQAEEDVEKSQWNYPEHPGGDCLPGAHHLQKHPTPSPWLDQAHEIWQARPWXPVQGHRLCGRPGRHFQNGLHPKRWQWCQGVGSVQLPRSGVGMGMYNTDESISGFAHSCFQYAIQKKWPLYMSTKNTILKAYDGRFKDIFQEIFDKHYKTDFDKNKIWYEHRLIDDMVAQVLKSSGGFVWACKNYDGDVQSDILAQGFGSLGLMTSVLVCPDGKTIEAEAAHGTVTRHYREHQKGRPTSTNPIASIFAWTRGLEHRGKLDGNQDLIRFAQMLEKVCVETVESGAMTKDLAGCIHGLSNVKLNEHFLNTTDFL DTIKSNLDRALGRQ (SEQID NO:21 1); DPRLAVLLLGVQILVERWRLQWDHY YLCPHRVQAEEDVEKSQWNYPEH (SEQ IDNO:212); PGGDCLPGAHHLQKH PTPSPWLDQAHHHWQARPWXPVQGHRLCGRPGRHFQNGL (SEQ IDNO:213); HPKRWQWCQGVGSVQLPRSGVGMGMYN ESISGFAHSCFQYAIQKKWPLYMSTKNTILKAYDGRFKDIF (SEQ ID NO:214); QEIFDKHYKTDFDKNKIWYEHRLIDDMVAQVLKSSGGFVWACKNYDGDVQSDILAQGFGSLGL MTSVLVC (SEQ IDNO:215); PDGKTIEAEAAHGTVTRHYREHQKGRPTSTNPIASIFAWTRGLEHRGKLDGNQDLIRFAQMLEKVCVETV (SEQ ID NO:216); and/orESGAMTKDLAGCIHGLSNVKLNEHFLNTTDFLDTIKSNLDRALGRQ (SEQ ID NO:217).Polynucleotides encoding these polypeptides are also encompassed by theinvention.

This gene is expressed primarily in T-cells and B-cells, and to a lesserextent in breast cancer tissue.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, breast cancer, leukemia, HIV, andtonsillitis. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune/lymphoid system, and breast cancers, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., immune, reproductive, breast,cancerous and wounded tissues) or bodily fluids (e.g., lymph, breastmilk, serum, plasma, urine, synovial fluid and spinal fluid) or anothertissue or cell sample taken from an individual having such a disorder,relative to the standard gene expression level, i.e., the expressionlevel in healthy tissue or bodily fluid from an individual not havingthe disorder.

The tissue distribution in immune cells, combined with the detected gasbiological activity, and its homology to the NADH-isocitratedehydrogenase, suggests that polynucleotides and polypeptidescorresponding to this gene are useful for the treatment and/or diagnosisof diseases related to the proliferation of lymphoid cells. Furthermore,it could be especially useful in helping the body to produce maturelymphocytes to enhance the immune system. Protein, as well as,antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:54 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 2009 of SEQID NO:54, b is an integer of 15 to 2023, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:54, and whereb is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 45

This gene is expressed primarily in PHA treated T-cells.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, T-cell disorders. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types or cell types (e.g., immune, cancerousand wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,urine, synovial fluid and spinal fluid) or another tissue or cell sampletaken from an individual having such a disorder, relative to thestandard gene expression level, i.e., the expression level in healthytissue or bodily fluid from an individual not having the disorder.

The tissue distribution in T-cells suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the diagnosisand/or treatment of immune disorders involving T-cells. Furthermore, thegene product may play a role in the regulation of the proliferation;survival; differentiation; and/or activation of potentially allhematopoietic cell lineages, including blood stem cells. This geneproduct may be involved in the regulation of cytokine production,antigen presentation, or other processes that may also suggest ausefulness in the treatment of cancer (e.g., by boosting immuneresponses).

Since the gene is expressed in cells of lymphoid origin, the gene orprotein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues. Therefore it may be also used as an agent forimmunological disorders including arthritis, asthma, immune deficiencydiseases such as AIDS, leukemia, rheumatoid arthritis, inflammatorybowel disease, sepsis, acne, and psoriasis. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Expressionof this gene product in T cells also strongly suggests a role for thisprotein in immune function and immune surveillance. Protein, as well as,antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:55 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 871 of SEQID NO:55, b is an integer of 15 to 885, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:55, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 46

This gene is expressed primarily in fetal heart tissue.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, mitrovalve prolapse, congenitalheart diseases or arythmic heartbeats. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the cardiovascular system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types or cell types (e.g., cardiac,developmental, cancerous and wounded tissues) or bodily fluids (e.g.,lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in fetal heart suggests that polynucleotides andpolypeptides corresponding to this gene are useful for the treatmentand/or diagnosis of heart diseases especially in the developing fetus.Furthermore, the tissue distribution in fetal heart tissue indicatesthat the protein product of this gene is useful for the diagnosis,treatment, and/or prevention of conditions and pathologies of thecardiovascular system, such as heart disease, restenosis,atherosclerosis, stoke, angina, thrombosis, and wound healing. Thesequence itself could be used in genetic therapy, in utero, to correctdefects in the developing fetus. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:56 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 1092 of SEQID NO:56, b is an integer of 15 to 1106, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO: 56, andwhere b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 47

This gene is expressed primarily in spleen tissue of chronic lymphocyticleukemia patient.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, leukemia. Similarly, polypeptidesand antibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels may be routinely detected incertain tissues or cell types (e.g., immune, cancerous and woundedtissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in spleen tissue suggests that polynucleotidesand polypeptides corresponding to this gene are useful for the treatmentand/or diagnosis of patients with leukemia. Expression of this geneproduct in spleen tissue suggests a role in the regulation of theproliferation; survival; differentiation; and/or activation ofpotentially all hematopoietic cell lineages, including blood stem cells.This gene product may be involved in the regulation of cytokineproduction, antigen presentation, or other processes that may alsosuggest a usefulness in the treatment of cancer (e.g., by boostingimmune responses).

Since the gene is expressed in cells of lymphoid origin, the gene orprotein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues. Therefore it may be also used as an agent forimmunological disorders including arthritis, asthma, immune deficiencydiseases such as AIDS, leukemia, rheumatoid arthritis, inflammatorybowel disease, sepsis, acne, and psoriasis. In addition, this geneproduct may have commercial utility in the expansion of stem cells andcommitted progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Protein, aswell as, antibodies directed against the protein may show utility as atumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:57 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 750 of SEQID NO:57, b is an integer of 15 to 764, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:57, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 48

This gene is expressed primarily in liver cancer tissue, and to a lesserextent in bone marrow, neutrophils, and CD34 cells.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, liver cancer and scerosis of theliver. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thehepatic and lymphatic systems, expression of this gene at significantlyhigher or lower levels may be routinely detected in certain tissues orcell types (e.g., liver, immune, hematopoiectic, cancerous and woundedtissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in cancerous liver tissue suggests thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and/or diagnosis of liver cancer, possibly before theonset of symptoms. Similarly, this gene would be useful for thedetection and treatment of liver disorders and cancers (e.g.,hepatoblastoma, jaundice, hepatitis, liver metabolic diseases andconditions that are attributable to the differentiation of hepatocyteprogenitor cells). Protein, as well as, antibodies directed against theprotein may show utility as a tumor marker and/or immunotherapy targetsfor the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:58 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 724 of SEQID NO:58, b is an integer of 15 to 738, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:58, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 49

This gene is expressed primarily in synovial hypoxia tissue.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, connective tissue and jointdisorders. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly ofconnective tissue, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., connective, skeletal, cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in synovial hypoxia tissue suggests thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and/or diagnosis of diseases of connective tissue,particularly synovia, including but not limited to inflammation,rheumatoid arthritis, osteoarthritis, and cartilage tears and physicalinjury. Furthermore, polynucleotides and polypeptides corresponding tothis gene are useful in the diagnosis or treatment of various autoimmunedisorders such as rheumatoid arthritis, lupus, scleroderma, anddermatomyositis as well as dwarfism, spinal deformation, and specificjoint abnormalities as well as chondrodysplasias (ie. spondyloepiphysealdysplasia congenita, farmilial osteoarthritis, Atelosteogenesis type II,metaphyseal chondrodysplasia type Schmid). Protein, as well as,antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:59 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 427 of SEQID NO:59, b is an integer of 15 to 441, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:59, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 50

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: MIMGYKSQKTFGLFDLXXVKGKTSVLEFDFWVQIPVASLLALWLNRLLNSVKWALKXCVIHSVAVNX (SEQ ID NO: 218). Polynucleotidesencoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in B-cell lymphoma tissue.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, B-cell lymphoma. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the immune system, expression of thisgene at significantly higher or lower levels may be routinely detectedin certain tissues or cell types (e.g., immune, cancerous and woundedtissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovialfluid and spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in B-cell lymphoma suggests that polynucleotidesand polypeptides corresponding to this gene are useful for the diagnosisand/or treatment of immune or hematopoietic disorders, particularlythose involving proliferative cells or tissues such as in cancers.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:60 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 770 of SEQID NO:60, b is an integer of 15 to 784, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:60, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 51

This gene is expressed primarily in synovial hypoxia tissue.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, connective tissue disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of connectivetissue, expression of this gene at significantly higher or lower levelsmay be routinely detected in certain tissues or cell types (e.g.,connective, skeletal, cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder.

The tissue distribution in synovial hypoxia tissue suggests thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and/or diagnosis of diseases of connective tissue,particularly synovia, including but not limited to inflammation,rheumatoid arthritis, osteoarthritis, and cartilage tears and physicalinjury. Furthermore, polynucleotides and polypeptides corresponding tothis gene are useful in the diagnosis or treatment of various autoimmunedisorders such as rheumatoid arthritis, lupus, scleroderma, anddermatomyositis as well as dwarfism, spinal deformation, jointabnormalities, and chondrodysplasias (ie. spondyloepiphyseal dysplasiacongenita, familial osteoarthritis, Atelosteogenesis type II,metaphyseal chondrodysplasia type Schmid). Protein, as well as,antibodies directed against the protein may show utility as a tumormarker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:61 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 526 of SEQID NO:61, b is an integer of 15 to 540, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:61, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 52

This gene is expressed primarily in synovial cells stimulated with IL-1and TNF, and also in neutrophils.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, inflammation of connective tissueand joints. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly ofconnective tissue, expression of this gene at significantly higher orlower levels may be routinely detected in certain tissues or cell types(e.g., connective, skeletal, cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in synovial cells suggests that polynucleotidesand polypeptides corresponding to this gene are useful for the treatmentand/or diagnosis of inflammation of connective tissues, particularly thesynovium, in diseases such as rheumatoid arthritis, sepsis, infection ofthe joint, and tissue damage from physical injury. Furthermore,polynucleotides and polypeptides corresponding to this gene are usefulin the diagnosis or treatment of various autoimmune disorders such asrheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well asdwarfism, spinal deformation, joint abnormalities, and chondrodysplasias(ie. spondyloepiphyseal dysplasia congenita, familial osteoarthritis,Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid).Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:62 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 590 of SEQID NO:62, b is an integer of 15 to 604, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:62, and where bis greater than or equal to a+14.

Features of Protein Encoded by Gene No: 53

In specific embodiments, polypeptides of the invention comprise thefollowing amino acid sequence: MKSFPSTYFKSSSFQNTKYQTGVISVLISYEIEYAAFYHLSCKITLPSSVSRNCFISEXLVASQCLDT (SEQ ID NO:219). Polynucleotides encodingthese polypeptides are also encompassed by the invention.

This gene is expressed primarily in frontal cortex tissue sampled froman epileptic patient.

Polynucleotides and polypeptides of the invention are useful as reagentsfor differential identification of the tissue(s) or cell type(s) presentin a biological sample and for diagnosis of diseases and conditionswhich include, but are not limited to, neural disorders, such asepilepsy. Similarly, polypeptides and antibodies directed to thesepolypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of thenervous system, expression of this gene at significantly higher or lowerlevels may be routinely detected in certain tissues or cell types (e.g.,brain, cancerous and wounded tissues) or bodily fluids (e.g., lymph,serum, plasma, urine, synovial fluid and spinal fluid) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution in frontal cortex tissue suggests thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and/or diagnosis of epilepsy and related braindisorders. Elevated expression of this gene product within the frontalcortex tissue of the brain suggests that it may be involved in neuronalsurvival; synapse formation; conductance; neural differentiation, etc.Such involvement may impact many processes, such as learning andcognition. It may also be useful in the treatment of suchneurodegenerative disorders as schizophrenia; ALS; or Alzheimer's.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:63 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a−b, where a is any integer between 1 to 738 of SEQID NO:63, b is an integer of 15 to 752, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:63, and where bis greater than or equal to a+14.

TABLE 1A 5′ NT of First Last ATCC NT 5′ NT 3′ NT 5′ NT First AA AA AAFirst Last Deposit SEQ Total of of of AA of SEQ of of AA of AA cDNA NO:Z ID NT Clone Clone Start Signal ID Sig Sig Secreted of Gene No. CloneID and Date Vector NO: X Seq. Seq. Seq. Codon Pep NO: Y Pep Pep PortionORF 1 HSIDU19 209244 Uni-ZAP XR 11 680 1 680 352 352 67 1 21 22 73 Sep.12, 1997 2 HPRSB76 209244 pBluescript 12 741 1 741 127 127 68 1 22 23 59Sep. 12, 1997 3 HTEIL66 209244 Uni-ZAP XR 13 619 1 619 123 123 69 1 2425 134 Sep. 12, 1997 4 HSNAY92 209244 Uni-ZAP XR 14 611 1 611 127 127 701 29 30 35 Sep. 12, 1997 5 HSABG21 209244 pBluescript 15 585 1 585 96 9671 1 24 25 125 Sep. 12, 1997 SK- 6 HSAXB32 209244 Uni-ZAP XR 16 1040 11040 97 97 72 1 36 37 51 Sep. 12, 1997 7 HPEAD48 209244 Uni-ZAP XR 17625 1 625 203 203 73 1 18 19 97 Sep. 12, 1997 8 HPVAB94 209244 Uni-ZAPXR 18 819 1 819 80 80 74 1 25 26 44 Sep. 12, 1997 9 HSAXB81 209244Uni-ZAP XR 19 782 1 782 143 143 75 1 20 21 47 Sep. 12, 1997 10 HSAYC21209244 Uni-ZAP XR 20 655 1 655 155 155 76 1 26 Sep. 12, 1997 11 HSLCU73209244 Uni-ZAP XR 21 798 1 798 7 7 77 1 22 23 41 Sep. 12, 1997 12HSSFZ70 209244 Uni-ZAP XR 22 646 1 646 212 212 78 1 22 23 22 Sep. 12,1997 13 HTEIP36 209244 Uni-ZAP XR 23 752 1 752 22 22 79 1 19 20 58 Sep.12, 1997 14 HYBAY77 209244 Uni-ZAP XR 24 815 60 815 157 157 80 1 44 4547 Sep. 12, 1997 15 HROAE78 209244 Uni-ZAP XR 25 878 1 878 132 132 81 116 17 52 Sep. 12, 1997 16 HSAVP17 209244 Uni-ZAP XR 26 850 1 850 69 6982 1 18 19 44 Sep. 12, 1997 17 HSIEA14 209244 Uni-ZAP XR 27 788 1 788141 141 83 1 22 23 60 Sep. 12, 1997 18 HSNAQ47 209244 Uni-ZAP XR 28 8381 838 80 80 84 1 21 22 253 Sep. 12, 1997 18 HSNAQ47 209244 Uni-ZAP XR 64848 1 848 85 85 120 1 21 22 24 Sep. 12, 1997 19 HODDN65 209244 Uni-ZAPXR 29 755 1 755 251 251 85 1 14 15 20 Sep. 12, 1997 20 HPEAD79 209244Uni-ZAP XR 30 813 1 813 51 51 86 1 15 16 41 Sep. 12, 1997 21 HRDED19209244 Uni-ZAP XR 31 513 1 513 75 75 87 1 20 21 47 Sep. 12, 1997 22HSAYS89 209244 Uni-ZAP XR 32 576 1 576 94 94 88 1 15 16 43 Sep. 12, 199723 HTODK73 209244 Uni-ZAP XR 33 1019 4 1019 43 43 89 1 23 24 59 Sep. 12,1997 24 HSVAM10 209244 Uni-ZAP XR 34 433 1 433 46 46 90 1 27 28 51 Sep.12, 1997 25 HSNBN57 209244 Uni-ZAP XR 35 642 1 642 198 198 91 1 20 21 31Sep. 12, 1997 26 HSVBD22 209244 Uni-ZAP XR 36 667 1 667 61 61 92 1 24 2535 Sep. 12, 1997 27 HSAWA27 209244 Uni-ZAP XR 37 654 1 654 319 319 93 129 30 49 Sep. 12, 1997 28 HSFAH43 209244 Uni-ZAP XR 38 731 1 731 191 19194 1 22 23 24 Sep. 12, 1997 29 HSPAA60 209244 pSport1 39 378 1 378 198198 95 1 45 46 46 Sep. 12, 1997 30 HFAEF57 209277 Uni-ZAP XR 40 642 1642 232 232 96 1 42 43 86 Sep. 18, 1997 31 HEGAH43 209277 Uni-ZAP XR 41442 1 442 29 29 97 1 20 21 111 Sep. 18, 1997 32 HAGDG59 209277 Uni-ZAPXR 42 1734 44 1717 124 124 98 1 18 19 300 Sep. 18, 1997 33 HNGBX63209277 Uni-ZAP XR 43 517 1 517 120 120 99 1 15 16 104 Sep. 18, 1997 34HE2AG50 209277 Uni-ZAP XR 44 486 1 486 19 19 100 1 32 33 43 Sep. 18,1997 35 HCUIN80 209277 ZAP Express 45 826 1 826 106 106 101 1 16 17 49Sep. 18, 1997 36 HADCL29 209277 pSport1 46 694 1 694 248 248 102 1 16 1747 Sep. 18, 1997 37 HAPPS89 209277 Uni-ZAP XR 47 856 1 856 54 54 103 129 30 99 Sep. 18, 1997 38 HFGAH44 209277 Uni-ZAP XR 48 1643 1 1643 34 34104 1 20 21 58 Sep. 18, 1997 39 HFIHZ96 209277 pSport1 49 709 1 709 3939 105 1 24 25 64 Sep. 18, 1997 40 HFIUR10 209277 pSport1 50 541 1 54150 50 106 1 22 23 44 Sep. 18, 1997 41 HLDNA86 209277 pCMVSport 51 720 1717 45 45 107 1 31 32 92 Sep. 18, 1997 3.0 42 HNGAN75 209277 Uni-ZAP XR52 979 1 979 41 41 108 1 25 26 25 Sep. 18, 1997 43 HCUIO20 209277 ZAPExpress 53 380 1 380 43 43 109 1 19 20 67 Sep. 18, 1997 44 HLTEF12209277 Uni-ZAP XR 54 2023 624 1498 686 686 110 1 21 22 44 Sep. 18, 199745 HCFBJ91 209277 pSport1 55 885 1 885 61 61 111 1 20 21 52 Sep. 18,1997 46 HHFHP90 209277 Uni-ZAP XR 56 1106 1 1106 42 42 112 1 14 15 43Sep. 18, 1997 47 HLYCQ48 209277 pSport1 57 764 1 764 58 58 113 1 40 4164 Sep. 18, 1997 48 HHLAB07 209277 pBluescript 58 738 1 738 108 108 1141 34 35 69 Sep. 18, 1997 SK- 49 HFOXE30 209277 pSport1 59 441 1 441 3838 115 1 18 19 53 Sep. 18, 1997 50 HBJEL68 209277 Uni-ZAP XR 60 784 1784 109 109 116 1 33 34 41 Sep. 18, 1997 50 HBJEL68 209277 Uni-ZAP XR 65769 1 769 111 111 121 1 26 Sep. 18, 1997 51 HFOXA73 209277 pSport1 61540 1 540 25 25 117 1 17 18 52 Sep. 18, 1997 51 HFOXA73 209277 pSport166 539 1 539 15 15 122 1 17 Sep. 18, 1997 52 HFIUR35 209277 pSport1 62604 1 604 42 42 118 1 31 32 70 Sep. 18, 1997 53 HFPDE86 209277 Uni-ZAPXR 63 752 1 752 300 300 119 1 16 Sep. 18, 1997

TABLE 1B AA Tissue Distribution SEQ Library code: count OMIM Gene cDNAClone Contig SEQ ID ORF ID (see Table IV for Cytologic Disease No: IDID: NO: X (From-To) NO: Y Predicted Epitopes Library Codes) BandReference(s): 1 HSIDU19 520372 11 352-573 67 Thr-27 to Arg-45. AR251: 6,AR310: 6, 6p22- 106300, AR186: 6, AR249: 5, p21.3 108800, AR263: 5,AR052: 5, 120290, AR265: 5, AR253: 5, 120290, AR292: 4, AR055: 4,120810, AR061: 4, AR224: 4, 120820, AR247: 3, AR194: 3, 142857, AR309:3, AR169: 3, 142858, AR225: 3, AR266: 3, 150270, AR312: 3, AR202: 3,167250, AR215: 3, AR033: 3, 170261, AR163: 3, AR053: 3, 177900, AR204:3, AR283: 3, 179450, AR162: 3, AR295: 3, 201910, AR259: 3, AR198: 2,217000, AR183: 2, AR277: 2, 222100, AR282: 2, AR164: 2, 233100, AR226:2, AR233: 2, 235200, AR245: 2, AR300: 2, 248611, AR299: 2, AR311: 2,256550, AR193: 2, AR221: 2, 256550, AR096: 2, AR184: 2, 600202, AR217:2, AR177: 2, 600261, AR237: 2, AR223: 2, 601868, AR231: 2, AR089: 2,602280, AR232: 2, AR238: 2, 602475 AR273: 2, AR197: 2, AR229: 2, AR192:2, AR175: 2, AR291: 2, AR185: 2, AR294: 2, AR264: 2, AR293: 2, AR313: 2,AR161: 2, AR284: 2, AR060: 2, AR227: 2, AR213: 2, AR214: 2, AR165: 1,AR269: 1, AR205: 1, AR271: 1, AR216: 1, AR267: 1, AR201: 1, AR243: 1,AR234: 1, AR182: 1, AR218: 1, AR222: 1, AR256: 1, AR298: 1, AR316: 1,AR039: 1, AR255: 1, AR258: 1, AR270: 1, AR219: 1 H0036: 1 2 HPRSB76526310 12 127-306 68 AR169: 5, AR282: 4, 15q11- 103581, AR253: 4, AR266:4, q13 146150, AR221: 3, AR198: 2, 176270, AR245: 2, AR295: 2, 218000,AR272: 2, AR285: 2, 227220, AR176: 2, AR225: 2, 601623, AR286: 2, AR289:2, 601800, AR300: 2, AR214: 1, 601889, AR287: 1, AR055: 1, 602117 AR182:1, AR199: 1, AR212: 1, AR269: 1, AR170: 1, AR178: 1, AR297: 1, AR161: 1,AR293: 1, AR162: 1 H0211: 1 and L0759: 1. 3 HTEIL66 520125 13 123-527 69Glu-32 to Asn-54, AR161: 7, AR162: 7, His-98 to Arg-106, AR176: 7,AR163: 7, Ser-126 to Ser-134. AR266: 6, AR181: 6, AR229: 6, AR180: 6,AR182: 6, AR060: 6, AR228: 6, AR183: 6, AR173: 5, AR165: 5, AR261: 5,AR309: 5, AR269: 5, AR164: 5, AR239: 5, AR257: 5, AR313: 5, AR233: 5,AR289: 5, AR166: 5, AR236: 4, AR275: 4, AR268: 4, AR300: 4, AR240: 4,AR255: 4, AR235: 4, AR277: 4, AR274: 4, AR267: 4, AR264: 4, AR177: 4,AR247: 4, AR238: 4, AR270: 4, AR217: 4, AR170: 4, AR214: 4, AR179: 4,AR089: 4, AR104: 4, AR175: 4, AR061: 4, AR224: 4, AR237: 4, AR283: 3,AR282: 3, AR291: 3, AR316: 3, AR096: 3, AR293: 3, AR299: 3, AR221: 3,AR196: 3, AR193: 3, AR296: 3, AR272: 3, AR262: 3, AR174: 3, AR295: 3,AR185: 3, AR231: 3, AR222: 3, AR178: 3, AR226: 3, AR230: 3, AR290: 3,AR312: 3, AR234: 3, AR227: 3, AR204: 3, AR053: 3, AR288: 3, AR191: 3,AR201: 3, AR258: 3, AR168: 3, AR311: 3, AR297: 3, AR215: 3, AR055: 3,AR246: 3, AR285: 2, AR287: 2, AR286: 2, AR294: 2, AR263: 2, AR232: 2,AR171: 2, AR250: 2, AR216: 2, AR243: 2, AR200: 2, AR203: 2, AR033: 2,AR190: 2, AR225: 2, AR189: 2, AR260: 2, AR199: 2, AR211: 2, AR256: 2,AR188: 2, AR212: 2, AR210: 1, AR172: 1, AR218: 1, AR219: 1, AR198: 1,AR252: 1 L0794: 7, L0758: 5 and H0038: 1. 4 HSNAY92 526730 14 127-234 70AR176: 3, AR282: 2, AR223: 2, AR204: 2, AR039: 2, AR221: 2, AR169: 2,AR053: 2, AR195: 2, AR205: 2, AR309: 2, AR264: 2, AR181: 2, AR291: 1,AR262: 1, AR270: 1, AR216: 1, AR183: 1, AR201: 1, AR269: 1, AR240: 1,AR224: 1, AR294: 1, AR179: 1, AR247: 1, AR198: 1 5 HSABG21 526366 15 96-473 71 Thr-32 to Leu-43. AR250: 8, AR171: 4, AR225: 4, AR263: 4,AR060: 3, AR282: 3, AR235: 2, AR221: 2, AR240: 2, AR261: 2, AR168: 2,AR283: 2, AR223: 2, AR178: 2, AR257: 1, AR104: 1, AR089: 1, AR170: 1,AR172: 1, AR185: 1, AR217: 1, AR277: 1, AR162: 1, AR214: 1, AR163: 1,AR199: 1, AR316: 1, AR096: 1, AR272: 1 T0039: 1 and L0751: 1. 6 HSAXB32520470 16 97-252 72 AR313: 48, AR089: 39, AR299: 30, AR316: 26, AR096:24, AR104: 20, AR185: 19, AR060: 17, AR277: 16, AR240: 11, AR161: 10,AR162: 10, AR163: 10, AR165: 9, AR164: 9, AR166: 8, AR275: 7, AR196: 6,AR283: 6, AR282: 6, AR053: 5, AR274: 5, AR312: 5, AR300: 4, AR173: 4,AR175: 4, AR257: 4, AR264: 4, AR211: 4, AR308: 4, AR309: 4, AR247: 4,AR174: 4, AR195: 4, AR213: 4, AR262: 3, AR178: 3, AR191: 3, AR236: 3,AR199: 3, AR189: 3, AR203: 3, AR200: 3, AR258: 3, AR261: 3, AR229: 3,AR286: 3, AR197: 3, AR297: 3, AR218: 3, AR242: 2, AR212: 2, AR233: 2,AR285: 2, AR295: 2, AR179: 2, AR255: 2, AR234: 2, AR230: 2, AR238: 2,AR288: 2, AR201: 2, AR243: 2, AR287: 2, AR176: 2, AR294: 2, AR177: 2,AR181: 2, AR204: 2, AR296: 2, AR215: 2, AR183: 2, AR182: 2, AR293: 2,AR266: 2, AR033: 2, AR272: 2, AR223: 2, AR225: 2, AR219: 2, AR217: 2,AR226: 2, AR237: 2, AR231: 2, AR269: 2, AR172: 2, AR228: 2, AR260: 2,AR235: 2, AR311: 1, AR193: 1, AR268: 1, AR239: 1, AR188: 1, AR227: 1,AR291: 1, AR289: 1, AR290: 1, AR216: 1, AR267: 1 S0114: 1 and L0608: 1.7 HPEAD48 520367 17 203-496 73 Gln-51 to Thr-61, AR196: 9, AR161: 9,Ser-65 to Thr-71, AR162: 9, AR163: 8, Pro-85 to Gln-91. AR173: 8, AR169:8, AR171: 8, AR313: 7, AR168: 7, AR223: 7, AR175: 6, AR263: 6, AR240: 6,AR096: 6, AR258: 6, AR180: 5, AR264: 5, AR261: 5, AR262: 5, AR257: 5,AR229: 5, AR176: 5, AR165: 5, AR300: 5, AR282: 5, AR164: 5, AR214: 5,AR269: 5, AR185: 5, AR275: 5, AR089: 5, AR166: 5, AR274: 4, AR270: 4,AR174: 4, AR199: 4, AR181: 4, AR217: 4, AR179: 4, AR191: 4, AR253: 4,AR247: 4, AR183: 4, AR234: 4, AR170: 4, AR266: 4, AR177: 4, AR299: 4,AR218: 4, AR236: 4, AR309: 4, AR312: 4, AR316: 4, AR238: 4, AR233: 4,AR235: 4, AR189: 4, AR215: 4, AR213: 4, AR311: 4, AR225: 3, AR224: 3,AR277: 3, AR293: 3, AR200: 3, AR212: 3, AR104: 3, AR033: 3, AR178: 3,AR226: 3, AR255: 3, AR230: 3, AR296: 3, AR268: 3, AR060: 3, AR203: 3,AR291: 3, AR188: 3, AR237: 3, AR172: 3, AR182: 3, AR285: 3, AR283: 3,AR198: 3, AR216: 2, AR192: 2, AR219: 2, AR228: 2, AR227: 2, AR295: 2,AR287: 2, AR267: 2, AR286: 2, AR260: 2, AR294: 2, AR239: 2, AR231: 2,AR297: 2, AR201: 2, AR290: 2, AR222: 2, AR053: 2, AR272: 2, AR190: 2,AR289: 2, AR288: 2, AR256: 2, AR204: 2, AR232: 2, AR207: 1, AR210: 1,AR055: 1, AR197: 1, AR193: 1, AR308: 1, AR205: 1 H0165: 1 8 HPVAB94526749 18  80-214 74 AR192: 5, AR242: 3, AR214: 3, AR195: 2, AR264: 2,AR168: 2, AR225: 2, AR277: 2, AR257: 1, AR172: 1, AR282: 1, AR171: 1,AR255: 1, AR275: 1, AR270: 1, AR296: 1, AR165: 1, AR182: 1, AR224: 1,AR295: 1 S0013: 1 9 HSAXB81 520471 19 143-286 75 AR313: 38, AR242: 24,AR300: 23, AR173: 23, AR204: 23, AR178: 22, AR180: 21, AR089: 21, AR163:21, AR162: 21, AR229: 21, AR247: 21, AR164: 21, AR166: 21, AR165: 20,AR161: 20, AR096: 20, AR175: 19, AR299: 18, AR258: 17, AR293: 17, AR197:17, AR269: 16, AR179: 16, AR270: 16, AR183: 16, AR176: 16, AR262: 16,AR257: 15, AR182: 15, AR218: 15, AR240: 15, AR181: 14, AR226: 14, AR296:14, AR268: 14, AR174: 14, AR234: 13, AR233: 13, AR312: 13, AR185: 13,AR238: 13, AR193: 12, AR245: 12, AR316: 12, AR201: 12, AR297: 11, AR230:11, AR264: 11, AR243: 11, AR231: 11, AR177: 11, AR294: 11, AR282: 11,AR192: 10, AR203: 10, AR237: 10, AR039: 10, AR196: 10, AR199: 10, AR236:10, AR267: 9, AR285: 9, AR060: 9, AR053: 9, AR266: 9, AR239: 9, AR191:9, AR033: 9, AR288: 9, AR198: 9, AR261: 9, AR228: 9, AR286: 8, AR287: 8,AR195: 8, AR205: 8, AR291: 8, AR255: 8, AR219: 8, AR274: 8, AR212: 8,AR277: 8, AR189: 7, AR253: 7, AR289: 7, AR252: 7, AR295: 7, AR250: 7,AR171: 7, AR213: 7, AR290: 7, AR260: 7, AR271: 7, AR275: 7, AR227: 7,AR104: 7, AR254: 7, AR256: 7, AR200: 6, AR207: 6, AR308: 6, AR309: 6,AR283: 6, AR172: 5, AR235: 5, AR232: 5, AR188: 5, AR061: 5, AR263: 5,AR169: 5, AR272: 5, AR168: 5, AR211: 5, AR055: 5, AR246: 4, AR190: 4,AR311: 3, AR170: 3, AR221: 2, AR210: 2, AR217: 2, AR222: 2, AR224: 2,AR225: 2, AR216: 2 S0114: 1 and H0542: 1. 10 HSAYC21 526188 20 155-23576 AR313: 10, AR242: 8, AR269: 8, AR089: 8, AR161: 7, AR165: 7, AR229:7, AR228: 7, AR162: 7, AR182: 7, AR164: 7, AR060: 7, AR163: 7, AR166: 7,AR096: 7, AR181: 7, AR233: 7, AR267: 6, AR299: 6, AR176: 6, AR238: 6,AR053: 6, AR239: 6, AR316: 6, AR196: 6, AR300: 6, AR257: 6, AR235: 5,AR236: 5, AR240: 5, AR268: 5, AR172: 5, AR177: 5, AR175: 5, AR173: 5,AR266: 5, AR309: 5, AR180: 5, AR191: 5, AR247: 5, AR237: 5, AR183: 5,AR179: 5, AR270: 5, AR226: 5, AR231: 5, AR104: 5, AR200: 4, AR168: 4,AR185: 4, AR262: 4, AR261: 4, AR255: 4, AR264: 4, AR204: 4, AR296: 4,AR253: 4, AR293: 4, AR282: 4, AR312: 4, AR192: 4, AR171: 4, AR258: 4,AR234: 4, AR189: 4, AR294: 4, AR230: 4, AR227: 4, AR190: 4, AR178: 4,AR055: 4, AR308: 4, AR285: 3, AR198: 3, AR212: 3, AR203: 3, AR174: 3,AR199: 3, AR291: 3, AR061: 3, AR277: 3, AR289: 3, AR287: 3, AR215: 3,AR193: 3, AR218: 3, AR207: 3, AR195: 3, AR290: 3, AR260: 3, AR272: 3,AR033: 3, AR283: 3, AR224: 3, AR201: 3, AR223: 3, AR288: 3, AR297: 3,AR286: 3, AR232: 3, AR243: 3, AR263: 2, AR225: 2, AR214: 2, AR256: 2,AR275: 2, AR219: 2, AR274: 2, AR188: 2, AR246: 2, AR295: 2, AR216: 2,AR210: 2, AR213: 2, AR217: 2, AR311: 2, AR271: 1, AR205: 1, AR211: 1,AR170: 1 S0114: 1, H0254: 1, H0637: 1 and L0783: 1. 11 HSLCU73 520237 21 7-132 77 AR173: 13, AR313: 13, AR229: 11, AR165: 10, AR242: 10, AR164:10, AR178: 10, AR161: 10, AR162: 10, AR166: 10, AR163: 10, AR180: 9,AR300: 9, AR175: 8, AR192: 8, AR262: 8, AR181: 8, AR247: 8, AR257: 8,AR179: 8, AR233: 7, AR198: 7, AR197: 7, AR226: 7, AR204: 7, AR258: 7,AR176: 7, AR254: 7, AR201: 7, AR234: 7, AR177: 7, AR293: 6, AR193: 6,AR183: 6, AR039: 6, AR238: 6, AR182: 6, AR218: 6, AR174: 6, AR245: 6,AR266: 6, AR264: 6, AR270: 6, AR312: 5, AR228: 5, AR261: 5, AR053: 5,AR275: 5, AR207: 5, AR239: 5, AR269: 5, AR274: 5, AR240: 5, AR299: 5,AR271: 5, AR230: 5, AR237: 5, AR185: 5, AR089: 5, AR231: 5, AR268: 5,AR243: 5, AR096: 4, AR250: 4, AR282: 4, AR195: 4, AR199: 4, AR297: 4,AR267: 4, AR255: 4, AR277: 4, AR236: 4, AR033: 4, AR212: 4, AR286: 4,AR263: 4, AR227: 4, AR205: 4, AR252: 4, AR296: 4, AR246: 4, AR219: 4,AR309: 4, AR213: 4, AR289: 4, AR294: 4, AR308: 3, AR188: 3, AR285: 3,AR191: 3, AR196: 3, AR200: 3, AR189: 3, AR295: 3, AR214: 3, AR203: 3,AR291: 3, AR232: 3, AR287: 3, AR216: 3, AR272: 3, AR316: 3, AR168: 3,AR260: 3, AR210: 3, AR223: 3, AR061: 3, AR288: 2, AR060: 2, AR290: 2,AR283: 2, AR256: 2, AR055: 2, AR104: 2, AR190: 2, AR217: 2, AR211: 2,AR172: 2, AR171: 2, AR311: 2, AR253: 2, AR221: 1 S0028: 1 12 HSSFZ70526499 22 212-280 78 Thr-17 to Leu-22. AR207: 24, AR263: 17, AR235: 15,AR195: 15, AR309: 14, AR197: 14, AR192: 13, AR201: 13, AR161: 13, AR162:13, AR308: 12, AR213: 12, AR198: 12, AR165: 12, AR164: 12, AR311: 12,AR166: 12, AR163: 12, AR261: 11, AR193: 11, AR060: 11, AR212: 11, AR053:11, AR089: 11, AR246: 11, AR177: 11, AR245: 11, AR039: 11, AR264: 10,AR252: 10, AR205: 10, AR170: 10, AR176: 9, AR239: 9, AR271: 9, AR282: 9,AR181: 9, AR253: 9, AR242: 9, AR223: 9, AR204: 9, AR061: 9, AR277: 9,AR312: 9, AR240: 9, AR055: 8, AR174: 8, AR229: 8, AR104: 8, AR033: 8,AR299: 8, AR214: 8, AR283: 8, AR233: 8, AR178: 8, AR316: 8, AR226: 7,AR236: 7, AR222: 7, AR227: 7, AR168: 7, AR217: 7, AR272: 7, AR169: 7,AR243: 7, AR228: 7, AR274: 7, AR250: 7, AR232: 7, AR231: 7, AR275: 6,AR182: 6, AR266: 6, AR185: 6, AR300: 6, AR171: 6, AR180: 6, AR238: 6,AR216: 6, AR179: 6, AR096: 6, AR262: 6, AR247: 6, AR183: 5, AR269: 5,AR313: 5, AR257: 5, AR196: 5, AR175: 5, AR270: 5, AR295: 5, AR254: 5,AR224: 5, AR234: 5, AR237: 5, AR230: 5, AR288: 5, AR255: 5, AR267: 5,AR225: 5, AR291: 5, AR268: 4, AR297: 4, AR172: 4, AR293: 4, AR258: 4,AR189: 4, AR188: 4, AR289: 4, AR191: 4, AR286: 4, AR199: 4, AR173: 4,AR287: 3, AR285: 3, AR296: 3, AR203: 3, AR200: 3, AR190: 3, AR294: 3,AR218: 3, AR211: 3, AR210: 2, AR290: 2, AR256: 2, AR221: 2, AR219: 2,AR260: 2, AR215: 2 H0135: 1 13 HTEIP36 520468 23  22-198 79 Glu-33 toArg-45. AR162: 8, AR161: 7, AR163: 7, AR235: 7, AR229: 6, AR183: 6,AR176: 6, AR173: 6, AR313: 5, AR178: 5, AR266: 5, AR309: 5, AR233: 5,AR165: 5, AR181: 5, AR257: 5, AR164: 5, AR182: 5, AR274: 5, AR166: 4,AR221: 4, AR275: 4, AR175: 4, AR264: 4, AR300: 4, AR228: 4, AR268: 4,AR261: 4, AR096: 4, AR269: 4, AR293: 4, AR262: 4, AR270: 4, AR267: 4,AR196: 4, AR089: 4, AR231: 4, AR291: 4, AR238: 4, AR177: 4, AR226: 4,AR247: 4, AR282: 3, AR255: 3, AR185: 3, AR239: 3, AR299: 3, AR234: 3,AR237: 3, AR179: 3, AR277: 3, AR174: 3, AR289: 3, AR188: 3, AR258: 3,AR236: 3, AR316: 3, AR199: 3, AR225: 3, AR060: 3, AR290: 3, AR250: 3,AR227: 3, AR203: 3, AR297: 3, AR191: 3, AR294: 3, AR285: 3, AR061: 3,AR218: 3, AR296: 3, AR230: 3, AR215: 3, AR217: 3, AR172: 3, AR055: 2,AR240: 2, AR286: 2, AR288: 2, AR287: 2, AR295: 2, AR189: 2, AR224: 2,AR308: 2, AR223: 2, AR200: 2, AR263: 2, AR232: 2, AR214: 2, AR033: 2,AR190: 2, AR260: 2, AR272: 2, AR312: 2, AR104: 2, AR222: 2, AR039: 2,AR171: 2, AR219: 2, AR212: 2, AR256: 2, AR311: 1, AR243: 1, AR213: 1,AR201: 1, AR195: 1, AR169: 1, AR211: 1, AR216: 1 H0038: 1 and H0616: 1.14 HYBAY77 520394 24 157-300 80 AR193: 6, AR204: 6, AR245: 5, AR243: 4,AR176: 4, AR162: 4, AR170: 4, AR165: 4, AR269: 4, AR250: 3, AR201: 3,AR161: 3, AR163: 3, AR246: 3, AR291: 3, AR164: 3, AR166: 3, AR197: 3,AR178: 3, AR221: 3, AR181: 3, AR282: 3, AR215: 3, AR271: 2, AR257: 2,AR266: 2, AR175: 2, AR168: 2, AR299: 2, AR264: 2, AR270: 2, AR262: 2,AR172: 2, AR055: 2, AR089: 2, AR226: 2, AR309: 2, AR312: 2, AR255: 2,AR293: 2, AR237: 2, AR277: 2, AR182: 2, AR225: 2, AR286: 2, AR217: 2,AR300: 2, AR228: 2, AR272: 2, AR231: 2, AR240: 2, AR258: 2, AR203: 2,AR233: 1, AR261: 1, AR290: 1, AR183: 1, AR268: 1, AR227: 1, AR239: 1,AR060: 1, AR232: 1, AR311: 1, AR296: 1, AR189: 1, AR216: 1, AR247: 1,AR191: 1, AR061: 1, AR294: 1, AR180: 1, AR177: 1, AR295: 1, AR188: 1,AR313: 1, AR179: 1, AR235: 1, AR104: 1, AR096: 1 H0318: 1 and H0041: 1.15 HROAE78 520365 25 132-290 81 Ser-14 to Gln-23, AR161: 5, AR162: 5,Pro-32 to Lys-39. AR163: 5, AR176: 5, AR309: 4, AR235: 4, AR275: 4,AR182: 4, AR266: 3, AR270: 3, AR239: 3, AR257: 3, AR168: 3, AR269: 3,AR267: 3, AR228: 3, AR169: 3, AR233: 3, AR181: 3, AR178: 3, AR236: 3,AR229: 3, AR255: 3, AR238: 3, AR216: 3, AR264: 3, AR179: 3, AR055: 3,AR242: 3, AR268: 3, AR231: 3, AR175: 3, AR180: 3, AR173: 3, AR286: 3,AR237: 3, AR164: 3, AR287: 2, AR177: 2, AR230: 2, AR225: 2, AR191: 2,AR247: 2, AR226: 2, AR224: 2, AR171: 2, AR295: 2, AR291: 2, AR166: 2,AR311: 2, AR222: 2, AR294: 2, AR288: 2, AR183: 2, AR282: 2, AR261: 2,AR300: 2, AR214: 2, AR290: 2, AR196: 2, AR227: 2, AR293: 2, AR221: 2,AR277: 2, AR061: 2, AR274: 2, AR297: 2, AR246: 2, AR188: 2, AR240: 2,AR223: 2, AR165: 2, AR285: 2, AR190: 2, AR289: 2, AR313: 2, AR312: 2,AR234: 2, AR200: 2, AR272: 2, AR096: 2, AR195: 2, AR203: 1, AR189: 1,AR089: 1, AR232: 1, AR258: 1, AR256: 1, AR262: 1, AR213: 1, AR217: 1,AR212: 1, AR210: 1, AR199: 1, AR174: 1, AR260: 1, AR316: 1, AR252: 1,AR193: 1, AR060: 1, AR219: 1 H0316: 1 16 HSAVP17 519846 26  69-203 82AR313: 28, AR089: 20, AR299: 16, AR316: 14, AR096: 14, AR185: 12, AR060:12, AR104: 11, AR277: 8, AR165: 8, AR242: 8, AR162: 7, AR161: 7, AR163:7, AR240: 7, AR166: 7, AR192: 7, AR245: 6, AR164: 6, AR173: 6, AR053: 5,AR264: 5, AR282: 5, AR269: 4, AR263: 4, AR274: 4, AR257: 4, AR300: 4,AR275: 4, AR193: 4, AR271: 3, AR312: 3, AR212: 3, AR283: 3, AR308: 3,AR247: 3, AR199: 3, AR205: 3, AR229: 3, AR261: 3, AR262: 3, AR235: 3,AR198: 3, AR196: 3, AR213: 2, AR178: 2, AR221: 2, AR207: 2, AR255: 2,AR311: 2, AR191: 2, AR243: 2, AR258: 2, AR176: 2, AR203: 2, AR168: 2,AR309: 2, AR183: 2, AR200: 2, AR204: 2, AR216: 2, AR189: 2, AR201: 2,AR172: 2, AR055: 2, AR253: 2, AR233: 1, AR246: 1, AR239: 1, AR195: 1,AR171: 1, AR238: 1, AR228: 1, AR222: 1, AR190: 1, AR289: 1, AR175: 1,AR188: 1, AR270: 1, AR297: 1, AR237: 1 S0114: 1 and H0402: 1. 17 HSIEA14520387 27 141-323 83 AR169: 5, AR039: 4, AR170: 3, AR172: 3, AR235: 3,AR183: 3, AR274: 3, AR246: 2, AR192: 2, AR282: 2, AR263: 2, AR288: 2,AR243: 2, AR171: 2, AR205: 2, AR175: 2, AR222: 2, AR217: 1, AR270: 1,AR283: 1, AR096: 1, AR053: 1, AR216: 1, AR201: 1, AR225: 1, AR289: 1,AR162: 1 H0036: 1 18 HSNAQ47 847453 28  80-838 84 Lys-84 to Lys-90,AR250: 5, AR060: 5, Phe-151 to Leu- AR274: 4, AR282: 3, 156, AR225: 3,AR254: 3, Ala-204 to Asp- AR104: 3, AR089: 3, 212, AR283: 3, AR299: 3,Ala-238 to Ala-245. AR096: 3, AR240: 2, AR316: 2, AR245: 2, AR224: 2,AR180: 2, AR313: 2, AR221: 2, AR185: 2, AR277: 2, AR275: 1, AR176: 1,AR257: 1, AR205: 1 L0771: 3, L0755: 2, S0358: 1, H0587: 1, L0471: 1,H0163: 1, L0529: 1, S0152: 1, H0521: 1 and S0436: 1. 18 HSNAQ47 52677964  85-159 120 19 HODDN65 520348 29 251-313 85 AR313: 26, AR268: 22,AR196: 21, AR173: 19, AR299: 17, AR229: 17, AR240: 16, AR300: 16, AR096:16, AR161: 15, AR162: 15, AR180: 15, AR175: 15, AR163: 15, AR178: 15,AR247: 14, AR258: 14, AR183: 14, AR168: 14, AR267: 13, AR181: 13, AR270:13, AR262: 13, AR257: 13, AR290: 12, AR171: 12, AR234: 12, AR174: 12,AR089: 12, AR199: 12, AR169: 12, AR238: 11, AR269: 11, AR218: 11, AR200:11, AR179: 11, AR293: 11, AR264: 11, AR177: 11, AR223: 11, AR236: 11,AR165: 11, AR185: 11, AR188: 10, AR228: 10, AR164: 10, AR182: 10, AR203:10, AR191: 10, AR275: 10, AR226: 10, AR166: 10, AR316: 10, AR233: 10,AR225: 10, AR189: 10, AR214: 9, AR237: 9, AR235: 9, AR213: 9, AR296: 9,AR176: 9, AR274: 9, AR261: 9, AR170: 9, AR231: 9, AR282: 9, AR255: 9,AR309: 8, AR277: 8, AR033: 8, AR053: 8, AR239: 8, AR060: 8, AR230: 8,AR294: 8, AR219: 8, AR253: 8, AR285: 8, AR260: 8, AR312: 8, AR297: 8,AR212: 7, AR263: 7, AR266: 7, AR222: 7, AR252: 7, AR291: 7, AR287: 7,AR288: 6, AR190: 6, AR039: 6, AR172: 6, AR254: 6, AR217: 6, AR286: 6,AR195: 6, AR256: 6, AR227: 6, AR308: 6, AR216: 5, AR215: 5, AR210: 5,AR295: 5, AR193: 5, AR242: 5, AR289: 5, AR245: 5, AR207: 5, AR198: 5,AR192: 5, AR104: 5, AR201: 5, AR272: 5, AR055: 5, AR271: 5, AR224: 4,AR232: 4, AR243: 4, AR311: 4, AR211: 4, AR061: 4, AR250: 4, AR283: 4,AR221: 4, AR246: 4, AR197: 4, AR205: 3, AR204: 3 H0328: 1 20 HPEAD79520202 30  51-176 86 Lys-16 to Ser-21, AR277: 24, AR176: 5, Gly-36 toAsp-41. AR039: 5, AR162: 5, AR205: 5, AR161: 5, AR235: 5, AR163: 5,AR282: 5, AR309: 5, AR168: 4, AR223: 4, AR228: 4, AR181: 4, AR266: 4,AR182: 4, AR269: 4, AR229: 4, AR257: 3, AR233: 3, AR272: 3, AR178: 3,AR180: 3, AR165: 3, AR264: 3, AR261: 3, AR268: 3, AR195: 3, AR164: 3,AR275: 3, AR166: 3, AR267: 3, AR183: 3, AR196: 3, AR238: 3, AR237: 3,AR236: 3, AR316: 3, AR171: 3, AR170: 3, AR179: 3, AR245: 3, AR060: 3,AR262: 3, AR193: 3, AR177: 3, AR255: 3, AR242: 3, AR289: 3, AR201: 3,AR230: 3, AR311: 3, AR254: 3, AR215: 2, AR290: 2, AR294: 2, AR204: 2,AR216: 2, AR231: 2, AR287: 2, AR299: 2, AR288: 2, AR227: 2, AR300: 2,AR033: 2, AR089: 2, AR191: 2, AR239: 2, AR173: 2, AR271: 2, AR225: 2,AR270: 2, AR295: 2, AR293: 2, AR296: 2, AR234: 2, AR185: 2, AR285: 2,AR214: 2, AR226: 2, AR274: 2, AR190: 2, AR203: 2, AR096: 2, AR199: 2,AR189: 2, AR247: 2, AR297: 2, AR200: 2, AR217: 2, AR246: 2, AR055: 2,AR175: 2, AR211: 2, AR232: 2, AR061: 2, AR283: 2, AR286: 2, AR053: 1,AR222: 1, AR263: 1, AR256: 1, AR260: 1, AR258: 1, AR210: 1, AR291: 1,AR188: 1, AR174: 1, AR312: 1, AR252: 1, AR224: 1, AR219: 1 H0165: 1 21HRDED19 526019 31  75-218 87 AR162: 3, AR163: 3, AR161: 3, AR096: 3,AR176: 3, AR204: 2, AR168: 2, AR165: 2, AR180: 2, AR282: 2, AR166: 2,AR217: 2, AR263: 2, AR221: 2, AR275: 2, AR236: 2, AR171: 2, AR214: 2,AR172: 2, AR264: 2, AR225: 1, AR288: 1, AR164: 1, AR272: 1, AR283: 1,AR274: 1, AR267: 1, AR246: 1, AR181: 1, AR199: 1, AR196: 1, AR233: 1,AR205: 1, AR285: 1, AR195: 1, AR257: 1, AR247: 1, AR089: 1 H0124: 1,L0809: 1 and H0134: 1. 22 HSAYS89 526621 32  94-225 88 AR060: 5, AR161:4, AR162: 4, AR163: 4, AR242: 4, AR221: 4, AR176: 3, AR235: 3, AR215: 3,AR309: 3, AR178: 3, AR283: 3, AR257: 3, AR104: 3, AR264: 3, AR165: 2,AR164: 2, AR270: 2, AR089: 2, AR166: 2, AR233: 2, AR236: 2, AR228: 2,AR289: 2, AR267: 2, AR277: 2, AR293: 2, AR230: 2, AR225: 2, AR238: 2,AR175: 2, AR181: 2, AR268: 2, AR262: 2, AR286: 2, AR196: 2, AR191: 2,AR269: 2, AR229: 2, AR294: 2, AR285: 2, AR237: 2, AR240: 2, AR316: 2,AR226: 2, AR290: 2, AR171: 2, AR179: 2, AR296: 2, AR188: 2, AR291: 2,AR172: 2, AR287: 2, AR227: 2, AR182: 2, AR239: 2, AR200: 1, AR185: 1,AR174: 1, AR231: 1, AR177: 1, AR039: 1, AR261: 1, AR234: 1, AR193: 1,AR183: 1, AR288: 1, AR190: 1, AR096: 1, AR195: 1, AR061: 1, AR300: 1,AR255: 1, AR258: 1, AR201: 1, AR189: 1, AR216: 1, AR204: 1, AR299: 1,AR211: 1 S0114: 1 23 HTODK73 526021 33  43-222 89 Gln-27 to Arg-36.AR180: 12, AR181: 9, 20 AR179: 9, AR240: 8, AR229: 8, AR178: 7, AR297:7, AR285: 7, AR235: 7, AR253: 6, AR291: 6, AR261: 6, AR204: 6, AR222: 5,AR221: 5, AR266: 5, AR288: 5, AR264: 5, AR163: 5, AR168: 5, AR309: 5,AR162: 5, AR161: 5, AR295: 5, AR214: 5, AR224: 5, AR212: 5, AR201: 5,AR300: 5, AR287: 5, AR293: 5, AR217: 5, AR223: 5, AR269: 5, AR255: 5,AR236: 5, AR311: 5, AR237: 5, AR296: 5, AR262: 5, AR183: 5, AR267: 5,AR182: 5, AR312: 5, AR290: 5, AR039: 4, AR216: 4, AR053: 4, AR215: 4,AR252: 4, AR225: 4, AR207: 4, AR172: 4, AR277: 4, AR171: 4, AR268: 4,AR165: 4, AR231: 4, AR254: 4, AR169: 4, AR230: 4, AR270: 4, AR170: 4,AR166: 4, AR286: 4, AR282: 4, AR173: 4, AR164: 4, AR289: 4, AR257: 4,AR200: 4, AR198: 4, AR175: 4, AR228: 4, AR271: 4, AR190: 4, AR177: 4,AR247: 4, AR263: 4, AR191: 4, AR205: 4, AR213: 4, AR260: 4, AR250: 4,AR233: 3, AR055: 3, AR308: 3, AR176: 3, AR188: 3, AR238: 3, AR243: 3,AR174: 3, AR234: 3, AR274: 3, AR294: 3, AR239: 3, AR226: 3, AR196: 3,AR061: 3, AR189: 3, AR299: 3, AR246: 3, AR199: 3, AR195: 3, AR219: 3,AR193: 3, AR313: 3, AR197: 3, AR211: 3, AR203: 3, AR275: 2, AR256: 2,AR089: 2, AR033: 2, AR232: 2, AR227: 2, AR316: 2, AR060: 2, AR210: 2,AR096: 2, AR258: 2, AR185: 2, AR272: 2, AR283: 2, AR218: 2, AR104: 1,AR192: 1 L0745: 7, L0771: 5, L0777: 3, L0803: 2, S0360: 1, H0351: 1,H0441: 1, H0052: 1, H0288: 1, H0424: 1, S0364: 1, H0634: 1, H0264: 1,H0494: 1, H0646: 1, L0372: 1, L0800: 1, L0764: 1, L0766: 1, L0774: 1,L0775: 1, L0805: 1, L5623: 1, L0788: 1, H0672: 1, L0746: 1, L0779: 1,L0757: 1 and S0436: 1. 24 HSVAM10 520328 34  46-201 90 AR313: 45, AR242:40, AR192: 36, AR173: 31, AR196: 28, AR204: 26, AR258: 26, AR300: 25,AR039: 25, AR240: 25, AR247: 24, AR096: 24, AR175: 23, AR089: 23, AR229:22, AR218: 22, AR262: 22, AR165: 21, AR185: 21, AR166: 21, AR174: 20,AR257: 20, AR179: 20, AR162: 20, AR164: 20, AR163: 20, AR161: 19, AR234:19, AR178: 19, AR199: 19, AR293: 18, AR269: 18, AR236: 18, AR299: 17,AR183: 17, AR233: 17, AR180: 17, AR198: 17, AR270: 17, AR193: 17, AR191:16, AR177: 16, AR181: 15, AR275: 15, AR226: 15, AR182: 15, AR268: 14,AR189: 14, AR243: 14, AR203: 14, AR238: 13, AR316: 13, AR176: 13, AR264:13, AR296: 13, AR231: 12, AR200: 12, AR312: 12, AR287: 12, AR285: 12,AR255: 11, AR294: 11, AR237: 11, AR060: 11, AR260: 11, AR261: 11, AR219:11, AR188: 11, AR297: 11, AR230: 11, AR205: 10, AR274: 10, AR235: 10,AR286: 10, AR197: 10, AR290: 9, AR207: 9, AR267: 9, AR266: 9, AR291: 9,AR201: 9, AR288: 9, AR295: 9, AR271: 9, AR282: 9, AR239: 9, AR195: 8,AR228: 8, AR033: 8, AR104: 8, AR277: 7, AR190: 7, AR212: 7, AR227: 7,AR263: 7, AR309: 7, AR245: 7, AR308: 6, AR213: 6, AR246: 6, AR053: 6,AR256: 5, AR289: 5, AR272: 5, AR232: 5, AR055: 4, AR061: 4, AR210: 4,AR211: 3, AR311: 3, AR283: 3, AR252: 2, AR253: 2, AR254: 2, AR250: 1,AR222: 1, AR171: 1 25 HSNBN57 526721 35 198-293 91 AR246: 3, AR217: 3,AR221: 3, AR192: 2, AR243: 2, AR171: 2, AR230: 2, AR205: 2, AR299: 2,AR161: 2, AR224: 2, AR309: 2, AR297: 2, AR089: 2, AR163: 2, AR213: 1,AR264: 1, AR257: 1, AR168: 1, AR181: 1, AR216: 1, AR222: 1, AR282: 1,AR170: 1, AR227: 1 H0163: 1 26 HSVBD22 520557 36  61-168 92 Pro-29 toSer-35. AR311: 48, AR225: 11, AR309: 7, AR176: 7, AR282: 6, AR197: 6,AR161: 6, AR162: 6, AR170: 6, AR266: 6, AR163: 5, AR228: 5, AR182: 5,AR178: 5, AR229: 4, AR267: 4, AR172: 4, AR261: 4, AR204: 4, AR181: 4,AR275: 4, AR177: 4, AR183: 4, AR165: 4, AR268: 4, AR269: 4, AR207: 4,AR233: 4, AR289: 4, AR271: 4, AR257: 4, AR164: 4, AR291: 4, AR193: 4,AR089: 4, AR238: 4, AR166: 4, AR230: 4, AR201: 3, AR237: 3, AR245: 3,AR061: 3, AR055: 3, AR175: 3, AR293: 3, AR198: 3, AR255: 3, AR239: 3,AR226: 3, AR060: 3, AR316: 3, AR231: 3, AR168: 3, AR221: 3, AR272: 3,AR294: 3, AR234: 3, AR262: 3, AR236: 3, AR179: 3, AR263: 3, AR274: 3,AR300: 3, AR270: 3, AR227: 3, AR299: 3, AR312: 3, AR288: 3, AR223: 3,AR171: 3, AR185: 3, AR174: 2, AR247: 2, AR104: 2, AR173: 2, AR039: 2,AR287: 2, AR285: 2, AR286: 2, AR295: 2, AR290: 2, AR224: 2, AR195: 2,AR296: 2, AR232: 2, AR203: 2, AR200: 2, AR033: 2, AR211: 2, AR096: 2,AR283: 2, AR191: 2, AR190: 2, AR189: 2, AR258: 2, AR277: 2, AR216: 2,AR214: 2, AR256: 2, AR196: 1, AR205: 1, AR199: 1, AR243: 1, AR222: 1,AR246: 1, AR264: 1, AR308: 1, AR188: 1, AR240: 1, AR313: 1, AR297: 1H0309: 1 27 HSAWA27 520302 37 319-465 93 Gly-31 to Phe-36. AR313: 65,AR089: 58, AR299: 47, AR316: 38, AR104: 34, AR096: 32, AR185: 29, AR060:27, AR277: 20, AR240: 19, AR196: 15, AR173: 13, AR311: 12, AR264: 12,AR309: 12, AR161: 11, AR162: 11, AR163: 11, AR207: 11, AR263: 10, AR282:10, AR213: 10, AR053: 9, AR257: 9, AR199: 9, AR242: 9, AR233: 9, AR283:9, AR261: 9, AR191: 8, AR300: 8, AR192: 8, AR165: 8, AR262: 8, AR195: 8,AR252: 8, AR308: 8, AR164: 8, AR275: 8, AR166: 8, AR189: 7, AR212: 7,AR312: 7, AR197: 7, AR203: 7, AR245: 7, AR247: 7, AR254: 7, AR198: 7,AR255: 6, AR200: 6, AR215: 6, AR258: 6, AR170: 6, AR188: 5, AR274: 5,AR238: 5, AR296: 5, AR193: 5, AR033: 5, AR221: 5, AR293: 5, AR179: 5,AR250: 5, AR246: 5, AR169: 5, AR229: 5, AR224: 4, AR271: 4, AR253: 4,AR175: 4, AR190: 4, AR177: 4, AR205: 4, AR216: 4, AR239: 4, AR234: 4,AR204: 3, AR236: 3, AR267: 3, AR287: 3, AR226: 3, AR272: 3, AR228: 3,AR201: 3, AR289: 3, AR268: 3, AR227: 3, AR297: 3, AR231: 3, AR217: 3,AR218: 3, AR176: 3, AR260: 3, AR055: 3, AR219: 2, AR237: 2, AR270: 2,AR210: 2, AR291: 2, AR256: 2, AR269: 2, AR172: 2, AR243: 2, AR214: 2,AR288: 2, AR168: 2, AR181: 2, AR183: 2, AR285: 2, AR222: 2, AR295: 2,AR171: 2, AR061: 1, AR225: 1, AR230: 1, AR294: 1, AR290: 1, AR039: 1S0114: 1 28 HSFAH43 520399 38 191-265 94 Asn-12 to Thr-18. AR196: 32,AR313: 22, AR165: 18, AR162: 18, AR161: 18, AR164: 18, AR089: 18, AR163:17, AR166: 17, AR181: 17, AR174: 16, AR175: 16, AR173: 14, AR178: 14,AR299: 14, AR182: 14, AR309: 13, AR053: 12, AR293: 12, AR177: 12, AR191:12, AR257: 11, AR226: 11, AR316: 11, AR300: 11, AR189: 11, AR236: 11,AR247: 11, AR261: 10, AR176: 10, AR312: 10, AR268: 10, AR258: 10, AR185:10, AR179: 10, AR096: 9, AR060: 9, AR212: 9, AR199: 9, AR264: 9, AR180:9, AR270: 9, AR183: 9, AR262: 9, AR240: 9, AR203: 8, AR296: 8, AR229: 8,AR200: 8, AR269: 8, AR266: 8, AR238: 8, AR308: 8, AR188: 8, AR235: 8,AR267: 8, AR239: 8, AR233: 7, AR275: 7, AR263: 7, AR197: 7, AR255: 7,AR274: 7, AR277: 7, AR285: 7, AR228: 7, AR295: 7, AR286: 7, AR104: 7,AR294: 7, AR297: 6, AR198: 6, AR254: 6, AR213: 6, AR218: 6, AR290: 6,AR190: 6, AR271: 6, AR288: 5, AR272: 5, AR230: 5, AR204: 5, AR287: 5,AR231: 5, AR289: 5, AR234: 5, AR291: 5, AR215: 5, AR237: 5, AR195: 5,AR260: 5, AR282: 5, AR192: 4, AR061: 4, AR172: 4, AR193: 4, AR232: 4,AR227: 4, AR033: 4, AR253: 4, AR219: 4, AR256: 4, AR205: 4, AR211: 4,AR201: 3, AR055: 3, AR242: 3, AR210: 3, AR225: 3, AR039: 3, AR283: 3,AR250: 3, AR311: 2, AR216: 2, AR246: 2 H0154: 1 29 HSPAA60 526447 39198-338 95 AR215: 5, AR204: 4, AR282: 4, AR224: 3, AR163: 3, AR162: 3,AR161: 3, AR168: 2, AR309: 2, AR270: 2, AR263: 2, AR311: 2, AR271: 2,AR269: 2, AR182: 2, AR225: 2, AR089: 2, AR176: 2, AR246: 2, AR201: 1,AR183: 1, AR181: 1, AR222: 1, AR212: 1, AR264: 1, AR165: 1, AR214: 1,AR164: 1, AR238: 1, AR178: 1, AR096: 1, AR171: 1, AR267: 1, AR228: 1,AR266: 1, AR294: 1, AR242: 1, AR199: 1, AR216: 1 L0805: 1 and H0478: 1.30 HFAEF57 534142 40 232-492 96 Leu-69 to Leu-74. AR241: 14, AR161: 14,AR162: 13, AR163: 13, AR313: 10, AR242: 10, AR201: 10, AR165: 9, AR164:9, AR252: 9, AR197: 9, AR194: 9, AR053: 9, AR166: 9, AR198: 8, AR245: 8,AR236: 8, AR192: 8, AR176: 8, AR206: 8, AR250: 8, AR212: 8, AR235: 8,AR196: 7, AR039: 7, AR271: 7, AR186: 7, AR052: 7, AR173: 7, AR204: 7,AR246: 7, AR253: 7, AR263: 7, AR207: 7, AR191: 7, AR275: 7, AR180: 7,AR226: 7, AR272: 7, AR247: 7, AR089: 6, AR181: 6, AR299: 6, AR195: 6,AR293: 6, AR244: 6, AR193: 6, AR312: 6, AR213: 6, AR229: 6, AR251: 6,AR188: 6, AR055: 6, AR202: 6, AR309: 6, AR287: 6, AR264: 6, AR238: 6,AR273: 6, AR300: 6, AR174: 6, AR177: 6, AR240: 6, AR257: 6, AR237: 5,AR243: 5, AR061: 5, AR233: 5, AR228: 5, AR261: 5, AR184: 5, AR182: 5,AR262: 5, AR185: 5, AR096: 5, AR270: 5, AR189: 5, AR190: 5, AR274: 5,AR248: 5, AR205: 5, AR183: 5, AR175: 5, AR288: 5, AR169: 5, AR033: 5,AR297: 5, AR269: 5, AR199: 5, AR178: 5, AR249: 5, AR295: 5, AR308: 4,AR223: 4, AR254: 4, AR060: 4, AR104: 4, AR216: 4, AR296: 4, AR282: 4,AR227: 4, AR290: 4, AR221: 4, AR266: 4, AR232: 4, AR239: 4, AR311: 4,AR179: 4, AR298: 4, AR200: 4, AR231: 4, AR285: 4, AR255: 4, AR268: 4,AR286: 4, AR267: 4, AR230: 4, AR316: 4, AR294: 4, AR214: 4, AR277: 4,AR234: 4, AR168: 4, AR258: 3, AR291: 3, AR170: 3, AR217: 3, AR203: 3,AR292: 3, AR171: 3, AR289: 3, AR310: 3, AR218: 3, AR265: 3, AR215: 3,AR259: 3, AR284: 3, AR283: 2, AR225: 2, AR222: 2, AR219: 2, AR260: 2,AR210: 2, AR172: 2, AR224: 2, AR211: 1, AR256: 1 S6024: 1 31 HEGAH43532596 41  29-364 97 Lys-35 to Glu-41, AR161: 7, AR163: 6, 20p13 192340,Ala-62 to Asn-67. AR162: 6, AR176: 6, 234200 AR263: 4, AR275: 4, AR269:4, AR266: 4, AR216: 4, AR214: 4, AR183: 4, AR192: 4, AR233: 4, AR235: 4,AR270: 4, AR267: 4, AR228: 4, AR309: 4, AR261: 3, AR172: 3, AR236: 3,AR272: 3, AR264: 3, AR182: 3, AR217: 3, AR288: 3, AR293: 3, AR257: 3,AR274: 3, AR178: 3, AR169: 3, AR245: 3, AR229: 3, AR255: 3, AR311: 3,AR268: 3, AR177: 3, AR294: 3, AR262: 3, AR166: 3, AR179: 3, AR175: 3,AR170: 3, AR224: 3, AR287: 3, AR164: 3, AR282: 3, AR238: 3, AR239: 3,AR171: 3, AR191: 3, AR237: 3, AR061: 3, AR300: 3, AR291: 3, AR221: 2,AR181: 2, AR234: 2, AR173: 2, AR196: 2, AR231: 2, AR240: 2, AR252: 2,AR285: 2, AR286: 2, AR190: 2, AR290: 2, AR168: 2, AR055: 2, AR174: 2,AR185: 2, AR289: 2, AR165: 2, AR308: 2, AR227: 2, AR295: 2, AR223: 2,AR232: 2, AR188: 2, AR297: 2, AR201: 2, AR256: 2, AR189: 2, AR104: 2,AR200: 2, AR247: 2, AR226: 2, AR060: 2, AR225: 2, AR089: 1, AR230: 1,AR312: 1, AR258: 1, AR211: 1, AR199: 1, AR277: 1, AR210: 1, AR316: 1,AR212: 1, AR180: 1, AR219: 1, AR203: 1, AR033: 1, AR299: 1, AR260: 1L0758: 5, H0550: 1, S0374: 1 and L0779: 1. 32 HAGDG59 534165 42 124-102698 Lys-29 to Val-34, AR039: 36, AR299:  4 Cys-94 to Asp-99, 24, AR251:24, AR206: Ser-102 to Val-107, 23, AR205: 21, AR248: Gln-133 to Lys- 20,AR252: 20, AR244: 139. 19, AR238: 18, AR186: 18, AR254: 16, AR263: 14,AR207: 14, AR250: 14, AR275: 13, AR249: 13, AR264: 13, AR246: 12, AR181:12, AR241: 12, AR204: 11, AR274: 11, AR269: 11, AR202: 11, AR185: 10,AR253: 10, AR243: 10, AR292: 10, AR052: 9, AR265: 9, AR310: 9, AR060: 9,AR316: 9, AR309: 9, AR191: 9, AR190: 9, AR273: 8, AR161: 8, AR268: 8,AR162: 8, AR270: 8, AR189: 8, AR163: 8, AR240: 8, AR053: 8, AR312: 8,AR089: 8, AR226: 8, AR096: 7, AR033: 7, AR290: 7, AR183: 7, AR237: 7,AR194: 7, AR177: 7, AR198: 7, AR174: 7, AR313: 7, AR201: 7, AR271: 7,AR104: 7, AR192: 7, AR175: 6, AR272: 6, AR213: 6, AR291: 6, AR239: 6,AR179: 6, AR235: 6, AR165: 6, AR061: 6, AR296: 6, AR308: 6, AR164: 5,AR267: 5, AR188: 5, AR284: 5, AR227: 5, AR166: 5, AR298: 5, AR176: 5,AR266: 5, AR178: 5, AR182: 5, AR234: 5, AR212: 5, AR295: 4, AR277: 4,AR193: 4, AR282: 4, AR300: 4, AR293: 4, AR232: 4, AR229: 4, AR285: 4,AR311: 4, AR196: 4, AR055: 4, AR231: 4, AR247: 4, AR173: 3, AR283: 3,AR184: 3, AR245: 3, AR233: 3, AR203: 3, AR197: 3, AR289: 3, AR257: 3,AR261: 3, AR294: 3, AR297: 2, AR242: 2, AR217: 2, AR288: 2, AR286: 2,AR218: 2, AR255: 2, AR195: 2, AR259: 2, AR256: 2, AR200: 2, AR219: 2,AR180: 2, AR228: 2, AR210: 2, AR199: 2, AR224: 1, AR211: 1, AR230: 1,AR236: 1, AR287: 1 S0422: 22, S0408: 9, L0659: 9, S0438: 8, S0354: 6,L0754: 6, S0126: 5, H0543: 5, S0358: 4, S0444: 4, S0406: 4, H0436: 4,L0740: 4, L0777: 4, H0144: 3, S0374: 3, L0750: 3, L0599: 3, H0170: 2,H0717: 2, H0740: 2, S0360: 2, S0410: 2, H0747: 2, H0749: 2, H0587: 2,H0574: 2, H0486: 2, H0575: 2, H0036: 2, S0003: 2, H0622: 2, L0475: 2,H0509: 2, L0667: 2, L0771: 2, L0662: 2, L0766: 2, L0804: 2, L0790: 2,H0710: 2, L0748: 2, L0745: 2, L0749: 2, L0731: 2, S0026: 2, H0422: 2,H0171: 1, H0686: 1, S0040: 1, H0716: 1, L0785: 1, L2991: 1, S0212: 1,S0442: 1, H0393: 1, L0717: 1, H0441: 1, H0497: 1, H0427: 1, H0590: 1,S0346: 1, S0474: 1, H0581: 1, H0746: 1, H0050: 1, H0239: 1, H0510: 1,H0266: 1, H0553: 1, H0169: 1, H0264: 1, H0494: 1, S0450: 1, S0440: 1,H0654: 1, H0652: 1, S0344: 1, H0529: 1, H0026: 1, L0371: 1, L0372: 1,L0764: 1, L0521: 1, L0768: 1, L0649: 1, L0652: 1, L0653: 1, L0809: 1,L0367: 1, L0663: 1, L0665: 1, S0428: 1, H0699: 1, H0547: 1, H0670: 1,H0660: 1, S0330: 1, S0378: 1, H0518: 1, H0521: 1, H0522: 1, S0028: 1,L0744: 1, L0439: 1, L0751: 1, S0031: 1, S0260: 1, L0581: 1, L0362: 1,H0136: 1, S0276: 1, H0506: 1 and H0721: 1. 33 HNGBX63 532615 43 120-43499 Pro-46 to Pro-53, AR170: 5, AR309: 3, His-55 to Cys-63. AR242: 3,AR282: 3, AR225: 3, AR193: 3, AR192: 3, AR214: 2, AR180: 2, AR172: 2,AR039: 2, AR311: 2, AR271: 2, AR053: 2, AR264: 2, AR300: 1, AR196: 1,AR178: 1, AR272: 1, AR277: 1, AR270: 1, AR237: 1, AR161: 1 S0052: 1 34HE2AG50 532595 44  19-150 100 Ser-37 to Tyr-43. AR252: 4, AR250: 4,AR289: 3, AR282: 3, AR169: 3, AR296: 3, AR180: 2, AR178: 2, AR183: 2,AR246: 2, AR168: 2, AR255: 2, AR185: 2, AR264: 2, AR213: 2, AR240: 2,AR309: 2, AR257: 2, AR233: 2, AR274: 1, AR215: 1, AR222: 1, AR216: 1H0170: 1, H0574: 1, H0052: 1, H0048: 1 and L0766: 1. 35 HCUIN80 53412845 106-255 101 Pro-26 to Arg-40, AR313: 9, AR173: 8, Pro-43 to Lys-49.AR161: 7, AR162: 7, AR163: 7, AR165: 5, AR164: 5, AR258: 5, AR236: 5,AR166: 5, AR089: 5, AR175: 5, AR196: 5, AR262: 5, AR096: 5, AR180: 5,AR264: 5, AR257: 4, AR247: 4, AR185: 4, AR240: 4, AR300: 4, AR299: 4,AR274: 4, AR263: 4, AR312: 4, AR177: 4, AR275: 4, AR218: 4, AR053: 4,AR234: 3, AR174: 3, AR191: 3, AR282: 3, AR229: 3, AR212: 3, AR293: 3,AR261: 3, AR269: 3, AR170: 3, AR199: 3, AR182: 3, AR179: 3, AR183: 3,AR277: 3, AR203: 3, AR060: 3, AR316: 3, AR198: 3, AR181: 3, AR200: 3,AR233: 3, AR311: 3, AR226: 3, AR238: 3, AR270: 3, AR286: 3, AR189: 3,AR294: 2, AR283: 2, AR104: 2, AR178: 2, AR296: 2, AR193: 2, AR285: 2,AR291: 2, AR219: 2, AR287: 2, AR289: 2, AR309: 2, AR237: 2, AR288: 2,AR225: 2, AR033: 2, AR295: 2, AR039: 2, AR231: 2, AR255: 2, AR172: 2,AR213: 2, AR176: 2, AR268: 2, AR197: 2, AR242: 2, AR260: 2, AR205: 2,AR195: 2, AR228: 2, AR230: 2, AR188: 2, AR297: 2, AR267: 1, AR266: 1,AR204: 1, AR227: 1, AR239: 1, AR290: 1, AR201: 1, AR252: 1, AR192: 1,AR190: 1, AR256: 1 H0402: 1 36 HADCL29 532056 46 248-391 102 AR215: 6,AR269: 3, AR253: 3, AR282: 3, AR053: 3, AR235: 3, AR172: 3, AR195: 3,AR170: 3, AR309: 2, AR264: 2, AR311: 2, AR225: 2, AR270: 2, AR266: 2,AR228: 2, AR296: 2, AR183: 2, AR237: 2, AR275: 2, AR268: 2, AR216: 2,AR230: 2, AR205: 1, AR199: 1, AR261: 1, AR181: 1, AR238: 1, AR166: 1,AR210: 1, AR231: 1, AR234: 1, AR313: 1, AR229: 1, AR274: 1, AR089: 1,AR277: 1, AR247: 1, AR061: 1, AR104: 1, AR096: 1 H0427: 1, L0748: 1 andL0754: 1. 37 HAPPS89 532135 47  54-353 103 AR313: 73, AR300: 52, AR192:52, AR161: 51, AR163: 51, AR162: 51, AR275: 51, AR264: 51, AR242: 44,AR274: 43, AR193: 42, AR240: 38, AR174: 35, AR053: 34, AR247: 33, AR185:33, AR229: 33, AR212: 33, AR312: 31, AR177: 31, AR165: 31, AR195: 31,AR196: 30, AR233: 30, AR164: 30, AR166: 29, AR234: 29, AR270: 28, AR269:28, AR213: 28, AR238: 27, AR254: 27, AR096: 26, AR181: 26, AR198: 25,AR263: 25, AR237: 24, AR236: 24, AR179: 23, AR089: 23, AR039: 23, AR226:22, AR268: 21, AR207: 21, AR230: 21, AR173: 20, AR239: 20, AR191: 20,AR308: 20, AR204: 19, AR183: 18, AR250: 18, AR104: 18, AR197: 18, AR277:17, AR180: 17, AR033: 17, AR189: 16, AR253: 16, AR252: 16, AR311: 16,AR175: 16, AR235: 16, AR228: 15, AR257: 15, AR182: 15, AR272: 15, AR178:15, AR231: 15, AR267: 15, AR282: 15, AR201: 15, AR293: 14, AR199: 14,AR271: 14, AR188: 13, AR299: 13, AR227: 13, AR245: 13, AR176: 13, AR243:12, AR262: 12, AR258: 12, AR203: 12, AR287: 11, AR296: 11, AR316: 11,AR286: 11, AR309: 11, AR200: 11, AR060: 10, AR190: 10, AR285: 10, AR290:10, AR291: 10, AR266: 10, AR205: 9, AR288: 9, AR297: 9, AR232: 9, AR294:9, AR218: 8, AR261: 7, AR289: 7, AR246: 7, AR169: 6, AR283: 6, AR295: 6,AR255: 6, AR219: 6, AR055: 5, AR171: 5, AR061: 5, AR223: 4, AR260: 4,AR168: 4, AR222: 3, AR170: 3, AR217: 3, AR216: 3, AR256: 3, AR214: 2,AR224: 2, AR225: 2, AR172: 2, AR211: 2, AR210: 2, AR215: 1 H0575: 1 38HFGAH44 533132 48  34-210 104 Pro-30 to Gln-35, AR235: 7, AR269: 7,Pro-44 to Leu-50. AR181: 6, AR257: 5, AR161: 5, AR162: 5, AR236: 5,AR178: 5, AR165: 5, AR182: 5, AR163: 5, AR287: 5, AR164: 5, AR291: 5,AR255: 5, AR191: 5, AR166: 5, AR297: 5, AR261: 5, AR179: 5, AR173: 5,AR228: 5, AR176: 5, AR290: 5, AR172: 4, AR285: 4, AR293: 4, AR267: 4,AR183: 4, AR268: 4, AR294: 4, AR270: 4, AR309: 4, AR266: 4, AR264: 4,AR240: 4, AR215: 4, AR262: 4, AR231: 4, AR289: 4, AR295: 4, AR224: 4,AR180: 4, AR233: 4, AR174: 4, AR177: 4, AR096: 4, AR238: 4, AR175: 4,AR247: 4, AR308: 4, AR169: 4, AR272: 3, AR296: 3, AR313: 3, AR200: 3,AR196: 3, AR237: 3, AR286: 3, AR229: 3, AR258: 3, AR316: 3, AR216: 3,AR260: 3, AR300: 3, AR199: 3, AR188: 3, AR190: 3, AR275: 3, AR225: 3,AR234: 3, AR312: 3, AR168: 3, AR217: 3, AR223: 3, AR060: 3, AR221: 3,AR288: 3, AR189: 3, AR089: 3, AR299: 3, AR033: 3, AR230: 3, AR239: 3,AR210: 3, AR246: 3, AR039: 2, AR226: 2, AR243: 2, AR061: 2, AR104: 2,AR277: 2, AR170: 2, AR055: 2, AR282: 2, AR203: 2, AR227: 2, AR185: 2,AR274: 2, AR222: 2, AR171: 2, AR219: 2, AR250: 2, AR311: 2, AR254: 2,AR232: 2, AR214: 2, AR253: 2, AR213: 1, AR256: 1, AR218: 1, AR283: 1,AR211: 1, AR271: 1 L0439: 6, L0758: 5, L0766: 3, L0803: 3, L0754: 3,L0749: 3, S0010: 2, H0616: 2, L0775: 2, L0438: 2, L0777: 2, H0543: 2,H0656: 1, H0581: 1, H0178: 1, H0051: 1, H0647: 1, L0761: 1, L0521: 1,L0794: 1, L0784: 1, L0653: 1, L0809: 1, L0791: 1, L0663: 1, L0664: 1,H0690: 1, H0658: 1, H0696: 1, L0780: 1 and L0755: 1. 39 HFIHZ96 53204649  39-233 105 Asn-30 to Gly-37. AR221: 4, AR170: 3, AR296: 3, AR183: 3,AR163: 2, AR176: 2, AR282: 2, AR240: 2, AR224: 2, AR225: 2, AR297: 2,AR270: 2, AR180: 2, AR274: 2, AR104: 2, AR222: 2, AR264: 2, AR228: 2,AR232: 2, AR213: 2, AR229: 2, AR311: 1, AR313: 1, AR312: 1, AR230: 1,AR247: 1, AR238: 1, AR089: 1, AR096: 1, AR286: 1, AR190: 1, AR252: 1,AR277: 1, AR171: 1 H0728: 1, L0643: 1 and S0194: 1. 40 HFIUR10 532060 50 50-184 106 Gln-31 to Pro-39. AR169: 4, AR165: 4, AR161: 3, AR163: 3,AR215: 3, AR162: 3, AR166: 3, AR246: 3, AR252: 3, AR313: 3, AR089: 3,AR311: 3, AR266: 2, AR270: 2, AR180: 2, AR261: 2, AR164: 2, AR224: 2,AR269: 2, AR096: 2, AR236: 2, AR289: 2, AR201: 2, AR297: 2, AR312: 2,AR205: 2, AR217: 2, AR255: 2, AR172: 2, AR240: 2, AR216: 2, AR183: 2,AR309: 2, AR173: 2, AR291: 2, AR176: 2, AR196: 2, AR295: 1, AR264: 1,AR225: 1, AR299: 1, AR033: 1, AR174: 1, AR257: 1, AR282: 1, AR060: 1,AR230: 1, AR178: 1, AR177: 1, AR316: 1, AR168: 1, AR243: 1, AR283: 1,AR268: 1, AR277: 1, AR189: 1, AR290: 1, AR247: 1, AR055: 1, AR308: 1,AR288: 1, AR300: 1, AR237: 1, AR185: 1 H0265: 2, L0591: 2, H0556: 1,S0356: 1, H0271: 1, H0622: 1, S0428: 1, S0434: 1 and S0196: 1. 41HLDNA86 535730 51  45-323 107 Arg-35 to Ala-41. AR186: 162, AR298: 16160, AR241: 158, AR259: 155, AR227: 154, AR233: 143, AR275: 136, AR055:135, AR232: 134, AR192: 131, AR061: 131, AR175: 130, AR284: 127, AR194:125, AR204: 120, AR295: 120, AR237: 117, AR285: 116, AR033: 112, AR286:111, AR300: 109, AR184: 108, AR185: 107, AR226: 107, AR238: 107, AR293:105, AR177: 100, AR198: 100, AR231: 100, AR299: 100, AR206: 99, AR202:97, AR271: 95, AR052: 94, AR273: 91, AR104: 84, AR243: 84, AR179: 83,AR292: 80, AR294: 77, AR291: 71, AR282: 70, AR229: 68, AR244: 67, AR283:67, AR267: 63, AR234: 63, AR053: 61, AR182: 60, AR310: 57, AR205: 57,AR039: 56, AR183: 56, AR274: 56, AR089: 52, AR060: 51, AR247: 49, AR296:48, AR251: 46, AR240: 46, AR269: 46, AR309: 45, AR316: 43, AR213: 43,AR258: 42, AR246: 39, AR289: 38, AR265: 38, AR218: 38, AR290: 37, AR268:36, AR270: 36, AR211: 35, AR219: 35, AR277: 34, AR256: 34, AR312: 33,AR161: 32, AR162: 32, AR228: 31, AR096: 29, AR163: 29, AR239: 29, AR266:29, AR248: 28, AR262: 27, AR203: 27, AR174: 27, AR199: 26, AR249: 25,AR230: 25, AR287: 23, AR195: 23, AR311: 22, AR191: 22, AR190: 22, AR263:21, AR236: 21, AR313: 21, AR288: 21, AR181: 20, AR253: 20, AR257: 20,AR196: 19, AR193: 19, AR188: 19, AR201: 19, AR210: 19, AR255: 17, AR176:17, AR200: 17, AR189: 17, AR264: 17, AR178: 17, AR272: 16, AR260: 15,AR261: 14, AR173: 14, AR297: 14, AR197: 14, AR212: 12, AR180: 12, AR165:11, AR164: 11, AR235: 11, AR166: 10, AR207: 10, AR254: 9, AR250: 9,AR221: 7, AR245: 7, AR216: 7, AR308: 6, AR242: 6, AR169: 6, AR215: 6,AR217: 5, AR252: 5, AR172: 4, AR225: 4, AR224: 4, AR223: 3, AR222: 3,AR214: 2, AR168: 2, AR171: 2, AR170: 1 L0747: 13, L0731: 10, L0758: 10,L0769: 7, L0740: 7, H0545: 6, L0803: 6, L0809: 6, L0755: 6, S0360: 5,L0775: 5, L0748: 5, L0749: 5, H0305: 4, S0358: 4, H0266: 4, S0126: 4,L0757: 4, H0556: 3, H0740: 3, H0722: 3, H0530: 3, S0438: 3, L0771: 3,L0774: 3, L0657: 3, L0782: 3, L0789: 3, S0192: 3, H0171: 2, H0265: 2,S0040: 2, T0049: 2, H0638: 2, S0418: 2, S0278: 2, H0574: 2, H0486: 2,S0280: 2, H0309: 2, H0046: 2, H0616: 2, H0652: 2, S0142: 2, L0770: 2,L0768: 2, L0794: 2, L0518: 2, L0666: 2, H0658: 2, S3014: 2, S0027: 2,L0743: 2, L0751: 2, L0756: 2, L0753: 2, L0759: 2, L0593: 2, S0194: 2,H0624: 1, H0149: 1, H0717: 1, H0295: 1, H0294: 1, S0218: 1, H0656: 1,S0298: 1, H0484: 1, H0661: 1, H0228: 1, S0356: 1, S0442: 1, S0408: 1,H0730: 1, H0747: 1, H0393: 1, H0549: 1, H0441: 1, H0586: 1, H0333: 1,T0040: 1, H0575: 1, H0318: 1, S0474: 1, H0196: 1, H0746: 1, L0738: 1,H0544: 1, H0041: 1, H0571: 1, H0123: 1, H0081: 1, H0620: 1, H0024: 1,H0510: 1, H0687: 1, S0314: 1, S0003: 1, S0022: 1, H0615: 1, H0622: 1,T0006: 1, H0644: 1, H0617: 1, H0673: 1, H0708: 1, H0135: 1, H0163: 1,H0634: 1, H0551: 1, T0067: 1, H0100: 1, H0561: 1, H0641: 1, S0144: 1,S0422: 1, H0695: 1, L0520: 1, L0763: 1, L0638: 1, L5565: 1, L0373: 1,L0800: 1, L0641: 1, L0644: 1, L0648: 1, L0766: 1, L0804: 1, L0375: 1,L0805: 1, L0653: 1, L0776: 1, L0659: 1, L0783: 1, L5622: 1, L0663: 1,L0665: 1, H0684: 1, H0651: 1, S0378: 1, H0518: 1, S0044: 1, H0555: 1,S3012: 1, S0390: 1, S0206: 1, L0741: 1, L0750: 1, H0445: 1, S0436: 1,S0026: 1, H0653: 1, H0543: 1, H0423: 1, H0422: 1, S0462: 1 and H0721: 1.42 HNGAN75 532613 52  41-118 108 AR180: 4, AR266: 4, AR263: 4, AR225: 4,AR176: 4, AR235: 3, AR162: 3, AR161: 3, AR163: 3, AR217: 3, AR181: 3,AR272: 3, AR221: 2, AR274: 2, AR193: 2, AR224: 2, AR288: 2, AR269: 2,AR309: 2, AR053: 2, AR290: 2, AR171: 2, AR228: 2, AR257: 2, AR178: 1,AR261: 1, AR262: 1, AR195: 1, AR277: 1, AR237: 1, AR293: 1, AR190: 1,AR255: 1, AR254: 1, AR227: 1, AR264: 1, AR231: 1, AR200: 1, AR199: 1,AR286: 1, AR170: 1, AR226: 1, AR179: 1, AR175: 1, AR182: 1, AR287: 1,AR289: 1, AR233: 1, AR268: 1 S0052: 1 43 HCUIO20 534220 53  43-246 109Ser-41 to Lys-49. AR266: 4, AR252: 4, AR263: 4, AR170: 3, AR282: 3,AR219: 3, AR225: 3, AR254: 3, AR264: 3, AR053: 2, AR294: 2, AR274: 2,AR221: 2, AR308: 2, AR233: 2, AR214: 2, AR299: 2, AR165: 2, AR180: 2,AR227: 2, AR239: 2, AR257: 2, AR164: 2, AR205: 2, AR272: 2, AR217: 2,AR039: 2, AR291: 1, AR166: 1, AR193: 1, AR261: 1, AR224: 1, AR312: 1,AR222: 1, AR199: 1, AR060: 1, AR311: 1, AR218: 1, AR089: 1, AR267: 1H0402: 1 44 HLTEF12 532354 54 686-820 110 Cys-31 to Trp-36. AR235: 39,AR261: 15q26.1 166800, 23, AR216: 23, AR266: 210900 21, AR217: 20,AR291: 20, AR286: 19, AR215: 19, AR283: 18, AR256: 16, AR289: 15, AR258:15, AR295: 15, AR236: 15, AR263: 14, AR214: 14, AR260: 14, AR257: 14,AR172: 13, AR264: 13, AR221: 12, AR285: 12, AR309: 12, AR170: 10, AR171:10, AR297: 10, AR188: 10, AR288: 9, AR053: 9, AR168: 9, AR262: 9, AR287:9, AR169: 9, AR294: 9, AR224: 8, AR255: 8, AR231: 8, AR165: 8, AR164: 8,AR055: 8, AR282: 7, AR166: 7, AR250: 7, AR201: 7, AR308: 7, AR293: 7,AR271: 7, AR222: 7, AR207: 6, AR197: 6, AR311: 6, AR272: 6, AR161: 6,AR162: 6, AR163: 6, AR245: 6, AR182: 6, AR246: 6, AR200: 6, AR312: 5,AR313: 5, AR296: 5, AR239: 5, AR232: 5, AR176: 5, AR198: 5, AR243: 5,AR199: 5, AR196: 5, AR300: 5, AR195: 5, AR212: 5, AR205: 5, AR203: 4,AR247: 4, AR225: 4, AR226: 4, AR254: 4, AR237: 4, AR233: 4, AR234: 4,AR253: 4, AR213: 4, AR268: 4, AR191: 4, AR183: 4, AR218: 4, AR228: 4,AR316: 4, AR060: 3, AR193: 3, AR061: 3, AR229: 3, AR269: 3, AR089: 3,AR267: 3, AR039: 3, AR277: 3, AR275: 3, AR177: 3, AR240: 3, AR173: 3,AR219: 3, AR230: 3, AR211: 3, AR274: 3, AR270: 3, AR189: 2, AR290: 2,AR192: 2, AR223: 2, AR238: 2, AR175: 2, AR096: 2, AR181: 2, AR174: 2,AR299: 2, AR033: 2, AR104: 2, AR178: 2, AR190: 2, AR210: 1, AR179: 1,AR185: 1, AR227: 1 H0617: 24, S0436: 12, S0410: 7, H0620: 7, H0706: 6,L0604: 6, L0774: 5, L0665: 5, H0341: 4, H0484: 4, H0574: 4, L0163: 4,S0440: 4, L5622: 4, L0666: 4, H0521: 4, L0439: 4, L0747: 4, S0358: 3,S0408: 3, S0046: 3, H0370: 3, L0622: 3, H0013: 3, S0010: 3, H0041: 3,H0009: 3, L0471: 3, H0012: 3, H0199: 3, S0051: 3, H0083: 3, H0644: 3,H0090: 3, T0041: 3, L0770: 3, L0769: 3, L0646: 3, L0519: 3, L0663: 3,L3826: 3, L0750: 3, L0777: 3, L0752: 3, L0755: 3, L0731: 3, S0434: 3,L0588: 3, H0542: 3, H0543: 3, L0615: 2, S0116: 2, H0255: 2, H0729: 2,H0735: 2, H0208: 2, H0619: 2, H0549: 2, L3816: 2, S0474: 2, H0581: 2,H0046: 2, H0024: 2, H0266: 2, H0553: 2, H0181: 2, H0606: 2, H0708: 2,S0366: 2, H0087: 2, S0038: 2, H0100: 2, H0494: 2, H0560: 2, S0144: 2,S0426: 2, L0763: 2, L0372: 2, L0764: 2, L0662: 2, L0655: 2, L0783: 2,H0144: 2, H0702: 2, H0520: 2, S0126: 2, S0380: 2, S0406: 2, L0742: 2,L0779: 2, L0759: 2, H0445: 2, L0596: 2, L0592: 2, L0599: 2, L0608: 2,L0601: 2, H0423: 2, H0422: 2, H0265: 1, S0040: 1, H0713: 1, H0717: 1,H0716: 1, H0740: 1, H0295: 1, H0294: 1, H0650: 1, H0657: 1, H0656: 1,S0180: 1, S0029: 1, H0483: 1, H0664: 1, H0306: 1, H0305: 1, S0418: 1,S0420: 1, S0442: 1, S0376: 1, S0444: 1, S0360: 1, H0675: 1, H0676: 1,H0637: 1, H0580: 1, H0742: 1, H0728: 1, H0733: 1, S0045: 1, S0476: 1,H0645: 1, H0431: 1, H0643: 1, H0632: 1, L0623: 1, H0486: 1, T0109: 1,S0280: 1, H0156: 1, H0599: 1, H0575: 1, H0618: 1, H0318: 1, S0049: 1,H0746: 1, H0052: 1, H0597: 1, H0545: 1, H0562: 1, H0172: 1, H0050: 1,H0154: 1, S0362: 1, H0018: 1, H0095: 1, H0373: 1, N0004: 1, T0010: 1,H0355: 1, H0099: 1, S6028: 1, S0312: 1, S0314: 1, L0483: 1, H0604: 1,H0213: 1, H0031: 1, H0111: 1, H0032: 1, S0364: 1, H0124: 1, H0551: 1,H0272: 1, S0112: 1, L0354: 1, L0370: 1, H0745: 1, S0438: 1, H0509: 1,S0344: 1, S0422: 1, L0598: 1, L0640: 1, L5566: 1, L0761: 1, L0374: 1,L0771: 1, L0773: 1, L0768: 1, L0775: 1, L0776: 1, L0629: 1, L0657: 1,L0384: 1, L0809: 1, L0530: 1, L0710: 1, H0698: 1, H0701: 1, H0703: 1,H0725: 1, H0723: 1, H0724: 1, L3811: 1, L3825: 1, H0547: 1, H0593: 1,H0435: 1, H0659: 1, H0658: 1, H0670: 1, H0660: 1, H0648: 1, L0355: 1,H0436: 1, H0626: 1, L0748: 1, L0593: 1, L0361: 1, S0011: 1, H0136: 1,H0216: 1, H0506: 1 and H0721: 1. 45 HCFBJ91 531960 55  61-219 111 AR313:19, AR096: 13, AR165: 12, AR247: 12, AR164: 12, AR163: 11, AR166: 11,AR161: 11, AR162: 11, AR173: 10, AR089: 10, AR240: 10, AR183: 9, AR300:9, AR180: 9, AR229: 9, AR299: 8, AR179: 8, AR293: 8, AR274: 8, AR178: 7,AR264: 7, AR296: 7, AR226: 7, AR175: 7, AR242: 7, AR262: 7, AR257: 7,AR185: 7, AR270: 7, AR234: 7, AR181: 7, AR312: 7, AR252: 7, AR174: 6,AR272: 6, AR196: 6, AR238: 6, AR263: 6, AR269: 6, AR258: 6, AR285: 6,AR261: 6, AR297: 6, AR275: 6, AR316: 6, AR199: 6, AR200: 6, AR268: 6,AR233: 5, AR214: 5, AR216: 5, AR039: 5, AR235: 5, AR282: 5, AR309: 5,AR182: 5, AR231: 5, AR060: 5, AR104: 5, AR237: 5, AR176: 5, AR215: 5,AR053: 5, AR287: 5, AR189: 4, AR171: 4, AR197: 4, AR204: 4, AR177: 4,AR271: 4, AR217: 4, AR283: 4, AR291: 4, AR213: 4, AR169: 4, AR230: 4,AR224: 4, AR294: 4, AR290: 4, AR311: 4, AR277: 4, AR033: 4, AR195: 4,AR254: 4, AR308: 4, AR218: 4, AR191: 4, AR239: 4, AR223: 4, AR205: 4,AR236: 4, AR170: 4, AR295: 4, AR286: 4, AR203: 4, AR267: 4, AR212: 4,AR228: 4, AR219: 4, AR193: 4, AR192: 3, AR288: 3, AR266: 3, AR289: 3,AR255: 3, AR243: 3, AR172: 3, AR188: 3, AR227: 3, AR246: 3, AR232: 3,AR260: 2, AR168: 2, AR222: 2, AR250: 2, AR201: 2, AR225: 2, AR256: 2,AR190: 2, AR061: 2, AR055: 2, AR210: 2, AR211: 1, AR253: 1 H0422: 1 46HHFHP90 534615 56  42-173 112 AR313: 36, AR089: 24, AR299: 23, AR242:21, AR060: 18, AR196: 17, AR185: 16, AR316: 16, AR193: 16, AR096: 15,AR240: 14, AR192: 13, AR180: 12, AR300: 12, AR162: 12, AR039: 12, AR173:12, AR175: 12, AR161: 12, AR104: 12, AR164: 12, AR163: 11, AR204: 11,AR165: 11, AR258: 11, AR197: 11, AR229: 11, AR166: 11, AR182: 11, AR277:10, AR247: 10, AR199: 10, AR183: 10, AR198: 10, AR293: 10, AR262: 9,AR270: 9, AR235: 9, AR269: 9, AR179: 9, AR191: 9, AR238: 9, AR236: 9,AR178: 9, AR233: 9, AR234: 9, AR181: 9, AR195: 9, AR264: 9, AR257: 8,AR201: 8, AR053: 8, AR253: 8, AR312: 8, AR174: 8, AR285: 8, AR212: 8,AR245: 8, AR218: 8, AR296: 8, AR226: 8, AR207: 8, AR283: 7, AR282: 7,AR176: 7, AR243: 7, AR294: 7, AR287: 7, AR252: 7, AR254: 7, AR297: 7,AR271: 7, AR200: 7, AR275: 7, AR268: 7, AR213: 6, AR177: 6, AR255: 6,AR237: 6, AR231: 6, AR219: 6, AR188: 6, AR260: 6, AR267: 6, AR261: 6,AR228: 6, AR286: 6, AR295: 6, AR189: 5, AR290: 5, AR203: 5, AR291: 5,AR033: 5, AR274: 5, AR239: 5, AR288: 5, AR266: 5, AR205: 5, AR190: 4,AR309: 4, AR263: 4, AR230: 4, AR308: 4, AR227: 4, AR256: 4, AR289: 4,AR250: 3, AR221: 3, AR232: 3, AR246: 3, AR216: 3, AR217: 3, AR061: 2,AR211: 2, AR055: 2, AR311: 2, AR172: 2, AR210: 2, AR225: 2, AR222: 2,AR272: 1, AR169: 1, AR214: 1, AR224: 1, AR223: 1, AR215: 1 H0050: 2 andH0645: 1. 47 HLYCQ48 527757 57  58-252 113 Pro-42 to Lys-49. AR313: 35,AR196: 23, AR161: 19, AR173: 19, AR165: 19, AR242: 19, AR162: 18, AR164:18, AR166: 18, AR089: 18, AR163: 18, AR247: 16, AR258: 16, AR300: 15,AR229: 15, AR183: 15, AR199: 15, AR240: 14, AR179: 14, AR096: 14, AR234:14, AR175: 14, AR299: 13, AR178: 13, AR180: 13, AR185: 13, AR172: 13,AR262: 13, AR192: 12, AR181: 12, AR238: 12, AR257: 11, AR200: 11, AR174:11, AR293: 11, AR191: 10, AR230: 10, AR312: 10, AR182: 10, AR270: 10,AR269: 10, AR177: 10, AR285: 10, AR233: 10, AR275: 10, AR193: 10, AR226:10, AR296: 9, AR316: 9, AR203: 9, AR236: 9, AR218: 9, AR189: 9, AR260:9, AR198: 9, AR060: 8, AR213: 8, AR277: 8, AR264: 8, AR297: 8, AR053: 8,AR039: 8, AR254: 8, AR231: 8, AR237: 8, AR195: 8, AR228: 8, AR268: 8,AR287: 7, AR294: 7, AR219: 7, AR286: 7, AR188: 7, AR204: 7, AR207: 7,AR261: 7, AR212: 6, AR033: 6, AR255: 6, AR239: 6, AR290: 6, AR282: 6,AR243: 6, AR176: 6, AR295: 6, AR227: 6, AR288: 5, AR274: 5, AR250: 5,AR256: 5, AR291: 5, AR309: 5, AR267: 5, AR104: 5, AR308: 5, AR245: 5,AR266: 5, AR201: 5, AR253: 4, AR289: 4, AR272: 4, AR190: 4, AR235: 4,AR232: 4, AR263: 4, AR197: 3, AR221: 3, AR205: 3, AR271: 3, AR216: 2,AR061: 2, AR225: 2, AR224: 1, AR311: 1, AR171: 1, AR223: 1, AR210: 1,AR211: 1 H0445: 1 48 HHLAB07 532602 58 108-317 114 AR225: 4, AR261: 4,AR282: 3, AR164: 3, AR172: 3, AR250: 3, AR264: 3, AR166: 3, AR163: 2,AR161: 2, AR207: 2, AR176: 2, AR204: 2, AR168: 2, AR217: 2, AR253: 2,AR270: 2, AR236: 2, AR181: 2, AR165: 2, AR300: 2, AR214: 2, AR266: 2,AR246: 2, AR263: 2, AR179: 2, AR183: 2, AR193: 1, AR228: 1, AR182: 1,AR267: 1, AR272: 1, AR233: 1, AR224: 1, AR221: 1, AR257: 1, AR178: 1,AR216: 1, AR287: 1, AR195: 1, AR277: 1, AR229: 1 S0116: 1, H0402: 1,T0091: 1 and S0052: 1. 49 HFOXE30 532120 59  38-199 115 AR217: 3, AR215:3, AR169: 3, AR180: 3, AR176: 3, AR272: 3, AR172: 3, AR166: 2, AR225: 2,AR183: 2, AR309: 2, AR277: 2, AR195: 2, AR096: 2, AR274: 2, AR165: 2,AR246: 2, AR271: 2, AR230: 2, AR181: 2, AR164: 2, AR287: 2, AR264: 2,AR173: 2, AR039: 2, AR295: 1, AR224: 1, AR228: 1, AR282: 1, AR257: 1,AR269: 1, AR089: 1, AR192: 1, AR299: 1, AR175: 1, AR178: 1, AR222: 1,AR179: 1, AR060: 1, AR171: 1, AR289: 1, AR216: 1, AR296: 1, AR193: 1,AR311: 1, AR261: 1, AR182: 1, AR239: 1, AR205: 1, AR200: 1, AR236: 1,AR237: 1, AR238: 1 S0276: 1 50 HBJEL68 531924 60 109-231 116 Arg-32 toGly-40. AR313: 12, AR161: 11, AR162: 11, AR163: 11, AR165: 10, AR164:10, AR166: 10, AR176: 9, AR182: 9, AR089: 8, AR229: 8, AR196: 8, AR269:8, AR181: 8, AR178: 7, AR257: 7, AR180: 7, AR238: 7, AR228: 7, AR060: 7,AR309: 7, AR275: 7, AR299: 7, AR247: 7, AR316: 7, AR233: 6, AR174: 6,AR173: 6, AR270: 6, AR183: 6, AR053: 6, AR293: 6, AR266: 6, AR200: 6,AR236: 6, AR226: 6, AR282: 6, AR175: 6, AR104: 6, AR240: 6, AR096: 6,AR300: 6, AR177: 6, AR191: 6, AR296: 6, AR189: 5, AR267: 5, AR274: 5,AR261: 5, AR264: 5, AR268: 5, AR185: 5, AR258: 5, AR262: 5, AR290: 5,AR237: 5, AR179: 5, AR199: 5, AR254: 5, AR312: 5, AR218: 5, AR277: 5,AR231: 5, AR239: 5, AR207: 5, AR234: 5, AR223: 5, AR168: 5, AR193: 5,AR039: 5, AR230: 4, AR235: 4, AR289: 4, AR204: 4, AR212: 4, AR198: 4,AR255: 4, AR285: 4, AR271: 4, AR227: 4, AR061: 4, AR297: 4, AR287: 4,AR308: 4, AR192: 4, AR288: 4, AR286: 4, AR242: 4, AR295: 4, AR203: 4,AR201: 4, AR188: 4, AR291: 4, AR225: 4, AR294: 4, AR195: 3, AR217: 3,AR055: 3, AR232: 3, AR219: 3, AR210: 3, AR214: 3, AR033: 3, AR190: 3,AR272: 3, AR283: 3, AR172: 3, AR224: 3, AR252: 3, AR245: 3, AR213: 3,AR256: 3, AR260: 3, AR216: 3, AR211: 2, AR250: 2, AR311: 2, AR222: 2,AR197: 2, AR263: 2, AR205: 1, AR170: 1, AR171: 1 S0222: 1 and H0318: 1.50 HBJEL68 531873 65 111-191 121 51 HFOXA73 850699 61  25-180 117 AR264:3, AR197: 3, AR274: 3, AR168: 2, AR291: 2, AR205: 2, AR283: 2, AR162: 2,AR163: 1, AR224: 1, AR161: 1, AR230: 1, AR240: 1, AR266: 1, AR190: 1,AR263: 1, AR191: 1, AR277: 1, AR178: 1, AR217: 1, AR257: 1, AR182: 1,AR295: 1 S0276: 1 51 HFOXA73 532079 66  15-68  122 52 HFIUR35 532062 62 42-254 118 Val-4 to Glu-9, AR176: 4, AR215: 3, Lys-42 to Lys-47. AR221:3, AR313: 3, AR165: 3, AR170: 3, AR264: 3, AR168: 3, AR161: 3, AR164: 3,AR162: 3, AR163: 3, AR235: 2, AR053: 2, AR275: 2, AR309: 2, AR270: 2,AR089: 2, AR178: 2, AR254: 2, AR295: 2, AR262: 2, AR236: 2, AR242: 2,AR285: 2, AR277: 2, AR190: 2, AR166: 2, AR216: 2, AR257: 2, AR237: 2,AR171: 2, AR172: 2, AR185: 1, AR177: 1, AR243: 1, AR271: 1, AR312: 1,AR240: 1, AR191: 1, AR258: 1, AR183: 1, AR196: 1, AR222: 1, AR308: 1,AR247: 1, AR316: 1, AR300: 1, AR299: 1, AR228: 1, AR189: 1, AR234: 1,AR272: 1, AR104: 1, AR180: 1, AR268: 1, AR294: 1, AR175: 1, AR233: 1,AR229: 1, AR179: 1, AR269: 1 L0748: 1, L0608: 1 and S0196: 1. 53 HFPDE86527728 63 300-350 119 AR313: 48, AR229: 26, AR300: 26, AR173: 25, AR096:23, AR258: 23, AR299: 22, AR164: 22, AR257: 22, AR165: 22, AR161: 21,AR162: 21, AR262: 21, AR180: 20, AR163: 20, AR247: 20, AR166: 20, AR175:20, AR178: 19, AR183: 18, AR179: 18, AR181: 18, AR218: 18, AR270: 18,AR234: 17, AR226: 17, AR196: 17, AR089: 17, AR269: 17, AR240: 16, AR238:16, AR185: 15, AR199: 14, AR174: 14, AR182: 14, AR191: 14, AR177: 13,AR268: 13, AR233: 13, AR293: 13, AR231: 13, AR296: 13, AR219: 13, AR312:12, AR230: 12, AR189: 12, AR237: 12, AR316: 12, AR203: 12, AR260: 11,AR255: 11, AR236: 11, AR264: 11, AR176: 11, AR261: 10, AR267: 10, AR282:10, AR060: 10, AR275: 10, AR053: 10, AR228: 10, AR266: 10, AR286: 9,AR285: 9, AR239: 9, AR188: 9, AR242: 9, AR200: 9, AR033: 8, AR297: 8,AR274: 8, AR256: 8, AR294: 8, AR193: 8, AR212: 7, AR277: 7, AR227: 7,AR309: 7, AR290: 7, AR213: 7, AR192: 6, AR190: 6, AR039: 6, AR308: 6,AR287: 6, AR232: 6, AR263: 6, AR250: 6, AR104: 6, AR295: 6, AR289: 6,AR291: 6, AR169: 5, AR288: 5, AR210: 5, AR272: 5, AR243: 5, AR253: 5,AR195: 4, AR211: 4, AR170: 4, AR283: 4, AR204: 4, AR311: 4, AR172: 4,AR235: 4, AR254: 3, AR225: 3, AR197: 3, AR205: 3, AR245: 3, AR252: 3,AR061: 3, AR201: 3, AR055: 3, AR271: 3, AR216: 2, AR217: 2, AR214: 2,AR246: 2, AR223: 2, AR207: 2, AR168: 2, AR171: 2, AR224: 1, AR198: 1S0222: 1 and L0682: 1.

TABLE 1C SEQ ID cDNA NO: CONTIG SEQ ID EXON Clone ID X ID: BAC ID: A NO:B From-To HSIDU19 11 520372 AL391194 220  1-83 166-317  639-11421280-1372 2309-2753 HSIDU19 11 520372 AL391194 221  1-262 HSIDU19 11520372 AL391194 222  1-768 HTEIL66 13 520125 AC067727 223  1-5072558-2661 2869-3237 HSAXB32 16 520470 AC002369 224  1-586 2559-26513329-3426 3756-5088 HSAXB32 16 520470 AC011957 225  1-126 HSAXB32 16520470 AC016655 226   1-1025 HSAXB32 16 520470 AC010419 227   1-1024HSAXB32 16 520470 AC027536 228  1-119 HSAXB32 16 520470 AC018548 229  1-1025 HSAXB32 16 520470 AP001973 230  1-102 HSAXB32 16 520470AC002369 231  1-228 HSAXB32 16 520470 AC016655 232  1-729 HSAXB32 16520470 AC016655 233  1-244 HSAXB32 16 520470 AC010419 234  1-732 HSAXB3216 520470 AC010419 235  1-244 HSAXB32 16 520470 AC018548 236  1-730HSAXB32 16 520470 AC018548 237  1-244 HPVAB94 18 526749 AF165147 238  1-1087 HPVAB94 18 526749 AF131217 239   1-1087 HSAXB81 19 520471AL353771 240   1-5816 6416-7049 8127-8227 8937-8991 10861-1099911231-11539 12714-12960 14741-15620 15735-16103 16263-16776 18121-1825819724-20346 20585-21454 22251-22431 22675-22937 23080-23487 23555-2394424043-26493 HSAXB81 19 520471 AC019213 241  1-880 HSAXB81 19 520471AL354721 242  1-880 HSAXB81 19 520471 AL353771 243  1-437 HSAXB81 19520471 AL353771 244  1-339 HSAXB81 19 520471 AC019213 245   1-58166416-6688 8132-8232 8942-8996 10872-11005 11237-11545 12720-12966HSAXB81 19 520471 AC019213 246  1-369 HSAXB81 19 520471 AL354721 247 1-369 HSAYC21 20 526188 AC019279 248  1-637 HSAYC21 20 526188 AC011097249  1-647 HSAYC21 20 526188 AC011097 250  1-324 HSAYC21 20 526188AC011097 251  1-281 HSLCU73 21 520237 AL138784 252  1-456 1098-14561870-2167 2841-2938 3718-4105 4803-5382 5830-5976 6102-6299 6733-68327071-7843 8487-9355  9898-10417 10629-11625 11647-11696 11704-1174111813-12296 12489-13208 15576-15724 18189-18478 18985-19068 21115-2126726441-26769 26782-27289 27493-27991 29015-29282 29428-29482 29508-2993329940-30050 30185-31063 31143-32602 33715-34300 34847-35835 36406-3671936786-36929 37366-37814 38984-39108 40994-41111 41559-43991 HSLCU73 21520237 AL138784 253  1-450 HSSFZ70 22 526499 AP002833 254  1-586 HSSFZ7022 526499 AP000839 255  1-585 HSSFZ70 22 526499 AP002833 256  1-639HSSFZ70 22 526499 AP000839 257  1-639 HTEIP36 23 520468 AC037454 258 1-37 2996-3803 HSAVP17 26 519846 AC010633 259   1-1273 HSAVP17 26519846 AC010633 260  1-484 HSAVP17 26 519846 AC010633 261  1-140 HSIEA1427 520387 AL133519 262  1-368 1447-1751 1777-2025 3643-4256 6078-62056273-6840 8298-8430 10016-10172 16541-16665 17747-17893 18161-1854718862-19340 19799-20268 23067-23841 23848-23950 26005-26779 28885-2899829777-29973 HSIEA14 27 520387 AL109655 263  1-775 HSIEA14 27 520387AL133519 264  1-353  402-1224 HSNAQ47 28 847453 AC024468 265  1-835HSNAQ47 28 847453 AL162612 266   1-3929 HSNAQ47 28 847453 AC018593 267  1-3928 HSNAQ47 28 847453 AC025992 268   1-3928 HSNAQ47 28 847453AC024468 269  1-269 2419-2560 3305-3786 4474-5582 5736-6454 6554-88139717-9903 HSNAQ47 28 847453 AL162612 270  1-379 HSNAQ47 28 847453AC018593 271  1-379 HSNAQ47 28 847453 AC025992 272  1-379 HODDN65 29520348 AC073052 273  1-753 HODDN65 29 520348 AC073052 274  1-101 HPEAD7930 520202 AL158846 275  1-811 HPEAD79 30 520202 AL158846 276  1-98HPEAD79 30 520202 AL158846 277  1-101 HRDED19 31 526019 AC021221 278 1-506 HRDED19 31 526019 AC040946 279  1-506 HSAYS89 32 526621 AC069204280  1-624 HSAYS89 32 526621 AC011319 281  1-624 HSAYS89 32 526621AC069204 282  1-146 HSVAM10 34 520328 AC021164 283  1-363 HSVAM10 34520328 AC021164 284  1-164 HSVAM10 34 520328 AC021164 285  1-177 HSNBN5735 526721 AC007263 286  1-629 HSNBN57 35 526721 AL157736 287  1-629HSNBN57 35 526721 AL357093 288  1-629 HSVBD22 36 520557 AL138996 289 1-600 HSVBD22 36 520557 AL138996 290  1-311 HSAWA27 37 520302 AL358073291  1-646 HSAWA27 37 520302 AL358353 292  1-646 HSAWA27 37 520302AC022243 293  1-646 HSAWA27 37 520302 AL358073 294  1-138 HSAWA27 37520302 AC022243 295  1-138 HSFAH43 38 520399 Z99758 296  1-432 1573-16572425-2702 2830-3126 3331-3848 4530-8774  8939-10624 26471-32522 HSFAH4338 520399 Z95400 297   1-2805 2807-3714 HSFAH43 38 520399 Z92542 298 1-728 3448-3710 6061-6275 HSFAH43 38 520399 U69729 299   1-2395 HSFAH4338 520399 AP001821 300   1-1750 HSFAH43 38 520399 AP000068 301   1-6128HSFAH43 38 520399 AL391071 302  1-29 2979-3837 HSFAH43 38 520399AL365475 303   1-5542  9722-10059 12469-12825 17209-18808 23300-2342028096-28311 33950-34377 36732-36948 45973-46071 46680-49375 HSFAH43 38520399 AL356802 304   1-6186 HSFAH43 38 520399 AL355052 305   1-6182HSFAH43 38 520399 AL354833 306  1-999 HSFAH43 38 520399 AL354751 307  1-6172 HSFAH43 38 520399 AL353812 308   1-6038 HSFAH43 38 520399AL162390 309  1-442 3536-4070  8479-10006 11103-11289 16064-1638118580-19340 21066-21597 HSFAH43 38 520399 AL157915 310   1-1074 HSFAH4338 520399 AL138810 311   1-2603 3747-3810 4195-4655 8673-949112027-15126 18182-18479 22522-22762 25837-25984 28990-29105 29310-2948231547-31981 37753-38223 42637-45774 HSFAH43 38 520399 AL135978 312  1-1943 HSFAH43 38 520399 AL133381 313   1-3135 HSFAH43 38 520399AL133370 314   1-1073 HSFAH43 38 520399 AL133304 315   1-1241 HSFAH43 38520399 AL132826 316   1-5842 HSFAH43 38 520399 AL121879 317   1-3114HSFAH43 38 520399 AL110502 318  1-386  396-2931 HSFAH43 38 520399AL109628 319   1-1450 HSFAH43 38 520399 AL079303 320  1-27  49-784HSFAH43 38 520399 AL078644 321  1-702  835-6171 HSFAH43 38 520399AL049875 322   1-1845 HSFAH43 38 520399 AL035700 323   1-2442 HSFAH43 38520399 AL035689 324   1-1474 HSFAH43 38 520399 AL034408 325  1-3371909-5834 HSFAH43 38 520399 AL033522 326   1-2391 HSFAH43 38 520399AL033403 327  1-444  466-2420 HSFAH43 38 520399 AL031650 328   1-617610689-11002 HSFAH43 38 520399 AL031387 329   1-5310 HSFAH43 38 520399AL024507 330   1-6096 HSFAH43 38 520399 AL022395 331  1-639 4341-6175HSFAH43 38 520399 AL022394 332   1-1723 HSFAH43 38 520399 AL022165 333 1-300 1441-7576 HSFAH43 38 520399 AL022069 334   1-4180 HSFAH43 38520399 AL021408 335   1-1600 HSFAH43 38 520399 AF207550 336   1-1946HSFAH43 38 520399 AF130343 337   1-2009 HSFAH43 38 520399 AF027390 338 1-799 HSFAH43 38 520399 AF011889 339  1-127  131-6009 HSFAH43 38 520399AF001905 340  1-895 HSFAH43 38 520399 AC022078 341   1-6035 12528-1271612739-13728 17358-17923 18326-18548 20169-20267 20945-21314 23341-2495127225-27416 35928-36115 36448-36827 39241-39566 40073-42103 42112-4231742448-44311 HSFAH43 38 520399 AC011327 342   1-4926 7254-7384 HSFAH43 38520399 AC009948 343   1-2807 2928-3084 3825-4520 4618-4772 5297-56015669-7057 7150-8193 8825-8961 9412-9822 HSFAH43 38 520399 AC009501 344 1-116 4875-5137 5347-6009 6286-6690 6808-7890 9458-9518 14225-1444728608-28803 30489-30789 35053-39752 45421-45698 HSFAH43 38 520399AC009289 345   1-6032 6037-6158  7763-11661 HSFAH43 38 520399 AC008583346   1-2169 HSFAH43 38 520399 AC008134 347   1-5956 HSFAH43 38 520399AC008041 348   1-3014 HSFAH43 38 520399 AC007744 349   1-1223 HSFAH43 38520399 AC007743 350   1-2579 HSFAH43 38 520399 AC007570 351  1-441 848-1703 2937-3091 4107-4235 5420-5837 6762-6878 7258-7362 7851-855211012-11366 11683-11945 11992-12100 14355-15033 16562-16726 18157-1832118716-18981 21960-22123 23221-23312 24248-24333 HSFAH43 38 520399AC007436 352  1-710 HSFAH43 38 520399 AC007358 353  1-916 HSFAH43 38520399 AC007276 354   1-1511 2538-2927 4237-4537 5726-6097 6190-69238701-8766 HSFAH43 38 520399 AC007000 355   1-2015 HSFAH43 38 520399AC006984 356  1-832 HSFAH43 38 520399 AC006556 357   1-5944 8943-923412643-13118 15612-15759 15811-15938 15947-17554 24155-24455 34509-36783HSFAH43 38 520399 AC006313 358   1-1650 HSFAH43 38 520399 AC006226 359 1-211 1700-3957 4145-6149 HSFAH43 38 520399 AC006195 360   1-5323HSFAH43 38 520399 AC006144 361  1-865 HSFAH43 38 520399 AC006084 362  1-6054 HSFAH43 38 520399 AC005740 363   1-3402 HSFAH43 38 520399AC005166 364   1-6073 7526-7932 7938-8353 11867-12326 13590-1400919843-20521 20684-21147 23387-25220 HSFAH43 38 520399 AC005164 365  1-6153 HSFAH43 38 520399 AC005146 366   1-2558 HSFAH43 38 520399AC005090 367   1-6048 21583-23065 HSFAH43 38 520399 AC005023 368  1-4659 5290-6014 HSFAH43 38 520399 AC003099 369   1-1750 HSFAH43 38520399 AC003075 370  1-841 HSFAH43 38 520399 AC002379 371   1-2312HSFAH43 38 520399 AC002060 372   1-1446 HSFAH43 38 520399 AC000120 373  1-6147 HSFAH43 38 520399 AB045359 374   1-1022 HSFAH43 38 520399AC023555 375   1-6203 HSFAH43 38 520399 AC011739 376   1-2587 HSFAH43 38520399 AL159175 377   1-1638 HSFAH43 38 520399 AL158158 378   1-1176 4708-10789 HSFAH43 38 520399 AC021201 379   1-1440 3030-3062 HSFAH43 38520399 AC011768 380   1-1733 1937-2229 2247-2452 2914-3752 4277-43304469-6688 7268-7948 HSFAH43 38 520399 AL359754 381  1-248 1315-18392740-6257 HSFAH43 38 520399 AL355474 382  1-357 1494-1815 2463-53085329-8041 HSFAH43 38 520399 AL139136 383   1-2735 HSFAH43 38 520399AC012207 384   1-6160 HSFAH43 38 520399 AC009266 385   1-551611501-13845 HSFAH43 38 520399 AC005000 386  1-772 1063-1418 1837-20982118-2246 2664-2705 2758-2890 3334-5823 HSFAH43 38 520399 AL355305 387  1-1431 3215-3361 4470-4666 4833-4908 5442-5931 6349-6723 7136-81448628-8720  9983-10102 11507-11655 13693-13817 13820-15838 16993-1717718554-18715 HSFAH43 38 520399 AL133385 388   1-3135 HSFAH43 38 520399AC012447 389   1-2238 HSFAH43 38 520399 AC010192 390   1-1031 HSFAH43 38520399 AC004958 391   1-6152 HSFAH43 38 520399 AP000409 392   1-22922296-3173 HSFAH43 38 520399 AL356259 393  1-844 HSFAH43 38 520399AL138814 394  1-217  401-3497 5172-5396 7866-8155 8596-9620 10961-11122HSFAH43 38 520399 AC025555 395   1-1656 HSFAH43 38 520399 AC025508 396  1-6052 HSFAH43 38 520399 AC022845 397  1-792 HSFAH43 38 520399AC012321 398  1-312 532-920 1441-2015 2738-4897 HSFAH43 38 520399AC012036 399   1-1435 HSFAH43 38 520399 AL356513 400   1-4544 4599-4917HSFAH43 38 520399 AL121933 401   1-1249 HSFAH43 38 520399 AC069443 402 1-153 4951-5981 HSFAH43 38 520399 AC068139 403   1-1702 HSFAH43 38520399 AC011435 404   1-6054 HSFAH43 38 520399 AC011153 405   1-2044HSFAH43 38 520399 AC006357 406   1-6191 HSFAH43 38 520399 AC011401 407  1-3406 HSFAH43 38 520399 AC023965 408   1-1086 HSFAH43 38 520399AL359271 409   1-1840 HSFAH43 38 520399 AL355075 410  1-716 HSFAH43 38520399 AL109804 411  1-811 HSFAH43 38 520399 AC023109 412  1-447 479-3690 3704-4456 HSFAH43 38 520399 AC012600 413   1-1190 HSFAH43 38520399 AC027552 414   1-1081 HSFAH43 38 520399 AP001645 415   1-2650HSFAH43 38 520399 AP001567 416   1-6410 HSFAH43 38 520399 AP001562 417  1-4260 HSFAH43 38 520399 AP000598 418  1-838 HSFAH43 38 520399AL356982 419   1-2358 HSFAH43 38 520399 AL354708 420  1-481  501-1170HSFAH43 38 520399 AL157899 421  1-738  792-6970 HSFAH43 38 520399AL157762 422   1-2317 2972-3275 HSFAH43 38 520399 AC024947 423   1-2765HSFAH43 38 520399 AC010210 424   1-6175 HSFAH43 38 520399 AC007849 425  1-6052 HSFAH43 38 520399 AC007338 426   1-1055 HSFAH43 38 520399AC022122 427   1-2439 HSFAH43 38 520399 AL031601 428   1-3441 HSFAH43 38520399 AC058811 429  1-176  769-4422 4453-4885 4939-5853 HSFAH43 38520399 AC022194 430  1-667 874-966 2107-7838 HSFAH43 38 520399 AC068393431   1-3748 HSFAH43 38 520399 AC025624 432   1-2446 HSFAH43 38 520399AC074250 433  1-59 248-396 5576-5669 7524-7687 8145-8533 8571-88349397-9870 11221-11322 11562-11960 12482-13656 16780-16900 17004-1710117108-17489 17592-18932 HSFAH43 38 520399 AC023412 434  1-609  774-48934947-5863 HSFAH43 38 520399 AC027116 435   1-1733 1937-2229 2247-24522914-3752 4277-4330 4469-6688 7268-7948 HSFAH43 38 520399 AC026494 436  1-1041 1050-3506 HSFAH43 38 520399 AC073118 437   1-2077 HSFAH43 38520399 AP001321 438   1-6041 HSFAH43 38 520399 AP000974 439  1-792HSFAH43 38 520399 AP000445 440   1-4775 HSFAH43 38 520399 AL355489 441  1-2350 HSFAH43 38 520399 AL161645 442   1-1620 HSFAH43 38 520399AL139109 443  1-198  203-6067 HSFAH43 38 520399 AL138682 444  1-217 401-3497 5172-5396 7866-8155 8602-9620 10961-11122 HSFAH43 38 520399AL133124 445   1-2174 HSFAH43 38 520399 AL031726 446  1-751 HSFAH43 38520399 AC078802 447   1-6052 HSFAH43 38 520399 AC073487 448  1-5872146-3842 3989-4201 5499-5529 6178-6728 7177-7679 8088-9978 10716-1097710981-11359 11459-11652 11709-12255 12352-12457 12485-12662 12775-1285312946-13797 13854-13972 14616-14764 15230-15403 15550-15610 15784-1587916027-16157 16273-16359 16716-16906 17040-17252 18205-18727 18898-1948720516-20784 21205-21286 21386-21515 25809-26225 HSFAH43 38 520399AC069525 449   1-5002 HSFAH43 38 520399 AC063920 450  1-93 143-4541232-6963 HSFAH43 38 520399 AC021652 451  1-667 874-966 2107-7838HSFAH43 38 520399 AC021329 452   1-1733 1937-2229 2257-2450 2914-37524277-4330 4728-4851 HSFAH43 38 520399 AC020656 453   1-2070 HSFAH43 38520399 AC018845 454   1-1055 HSFAH43 38 520399 AC018840 455   1-49597250-7380 HSFAH43 38 520399 AC018494 456  1-762 HSFAH43 38 520399AC016119 457  1-951 HSFAH43 38 520399 AC011432 458   1-1032 4529-51945642-5695 17014-22913 HSFAH43 38 520399 AC010798 459   1-1440 1543-8503HSFAH43 38 520399 AC007623 460  1-441  848-1703 2937-3091 4107-42355420-5837 6762-6878 7258-7362 7851-8552 11012-11366 11683-1194511992-12100 14355-15033 16562-16726 18157-18321 18716-18981 21960-2212323221-23312 24248-24333 HSFAH43 38 520399 AC007345 461  1-32  142-6020HSFAH43 38 520399 AC015521 462   1-2151 HSFAH43 38 520399 AC069153 463  1-1431 3215-3361 4470-4666 4833-4908 5442-5931 6349-6723 7136-81448628-8720  9983-10102 13246-13371 13390-13464 13683-13807 13810-1582816983-17167 18544-18705 HSFAH43 38 520399 AC015464 464   1-3481 HSFAH4338 520399 AC010736 465   1-2077 HSFAH43 38 520399 AC025802 466   1-2446HSFAH43 38 520399 AC018568 467   1-1645 HSFAH43 38 520399 AC074245 468 1-640  816-1328 1629-3899 4841-4907 6138-6199 6282-6407 HSFAH43 38520399 AC011823 469   1-2288 2442-3104 4477-4593 13213-13374 13769-1397715164-15429 15575-15735 15834-15933 16814-18957 19984-20104 20707-2079422341-22556 24065-24247 24773-24898 25233-25574 26907-27007 28468-2921629538-30106 30118-30602 30656-31302 31349-31375 31381-31559 36563-4190341914-42326 42427-42595 HSFAH43 38 520399 AC011123 470   1-4261 HSFAH4338 520399 AC013708 471   1-1912 1930-2850 3505-3808 HSFAH43 38 520399AC037472 472   1-1127 HSFAH43 38 520399 AC011086 473  1-865 HSFAH43 38520399 AL136327 474   1-1175 HSFAH43 38 520399 AC021850 475   1-2657HSFAH43 38 520399 Z92542 476  1-230 HSFAH43 38 520399 U69729 477  1-486HSFAH43 38 520399 U69729 478  1-141 HSFAH43 38 520399 AL391071 479 1-228 HSFAH43 38 520399 AL354833 480  1-457 HSFAH43 38 520399 AL354751481  1-349 HSFAH43 38 520399 AL354751 482  1-274 HSFAH43 38 520399AL162390 483  1-187 HSFAH43 38 520399 AL162390 484  1-202 HSFAH43 38520399 AL157915 485  1-602 HSFAH43 38 520399 AL157915 486  1-543 HSFAH4338 520399 AL138810 487  1-716 HSFAH43 38 520399 AL133370 488  1-602HSFAH43 38 520399 AL133370 489  1-543 HSFAH43 38 520399 AL132826 490 1-102 HSFAH43 38 520399 AL110502 491  1-529 HSFAH43 38 520399 AL078644492  1-326 HSFAH43 38 520399 AL035689 493  1-105 HSFAH43 38 520399AL035689 494  1-139 HSFAH43 38 520399 AL034408 495  1-519 HSFAH43 38520399 AL033403 496  1-130 820-951 1765-1857 5765-5858 6925-71878041-8110 8229-8343 9072-9146 9399-9769 10180-10631 11051-11151 HSFAH4338 520399 AL024507 497  1-134 HSFAH43 38 520399 AL022395 498   1-16911822-2256 3102-3417 3512-4108 4358-4629 4923-6465 6879-7193 12019-1209114772-14872 17599-18179 22036-22216 22839-23187 28010-28356 29023-2936332379-32844 HSFAH43 38 520399 AL022395 499  1-514 HSFAH43 38 520399AL022394 500  1-132 HSFAH43 38 520399 AL022165 501  1-172 5426-55777890-8007 8100-8182 10387-10472 HSFAH43 38 520399 AL022165 502  1-424HSFAH43 38 520399 AL022069 503  1-170 HSFAH43 38 520399 AF207550 504 1-54 170-273 5331-5457 5684-5983 5992-6112 6853-6948 7283-74987631-7747 8055-8170 8851-8931 9035-9130 9252-9308 9410-9511  9831-1017611703-12423 13776-14987 15189-15458 15854-16422 HSFAH43 38 520399AF130343 505  1-126 5612-5752 HSFAH43 38 520399 AC022078 506  1-828HSFAH43 38 520399 AC011327 507  1-433 HSFAH43 38 520399 AC009948 508 1-298 HSFAH43 38 520399 AC009948 509  1-651 HSFAH43 38 520399 AC008583510   1-1146 HSFAH43 38 520399 AC008134 511  1-174 HSFAH43 38 520399AC008041 512  1-392 HSFAH43 38 520399 AC007570 513  1-167 1801-18654645-4745 6582-7129 8325-8394 11657-11736 11991-12093 12184-1226912394-12473 14154-14418 14760-15011 15741-18861 HSFAH43 38 520399AC007000 514  1-387 HSFAH43 38 520399 AC007000 515  1-117 HSFAH43 38520399 AC006984 516  1-374 HSFAH43 38 520399 AC006144 517  1-280 HSFAH4338 520399 AC006084 518  1-165 HSFAH43 38 520399 AC006084 519  1-544HSFAH43 38 520399 AC005740 520  1-140 HSFAH43 38 520399 AC002379 521 1-370 HSFAH43 38 520399 AC002379 522  1-465 HSFAH43 38 520399 AC002060523  1-420 HSFAH43 38 520399 AC000120 524  1-247 HSFAH43 38 520399AL158158 525  1-617 1915-4051 4392-4708 6818-7286 HSFAH43 38 520399AC011768 526  1-457 593-900  922-1022 HSFAH43 38 520399 AL359754 527 1-227 HSFAH43 38 520399 AC012207 528  1-515 HSFAH43 38 520399 AL355305529  1-327 HSFAH43 38 520399 AC012447 530  1-88 HSFAH43 38 520399AC010192 531  1-287 HSFAH43 38 520399 AP000409 532  1-183 1203-13053252-3431 3478-5013 HSFAH43 38 520399 AL138814 533  1-543 HSFAH43 38520399 AC025508 534  1-189 HSFAH43 38 520399 AC022845 535  1-419 HSFAH4338 520399 AC012036 536  1-297 HSFAH43 38 520399 AL356513 537  1-2961293-1827 1881-2275 2436-2731 HSFAH43 38 520399 AL121933 538  1-100HSFAH43 38 520399 AC069443 539  1-84 HSFAH43 38 520399 AC069443 540 1-287 HSFAH43 38 520399 AC011435 541  1-544 HSFAH43 38 520399 AC006357542  1-360 HSFAH43 38 520399 AC011401 543  1-142 HSFAH43 38 520399AL359271 544  1-331 HSFAH43 38 520399 AL355075 545  1-551 HSFAH43 38520399 AC012600 546  1-165 HSFAH43 38 520399 AC012600 547  1-285 HSFAH4338 520399 AC027552 548  1-145 HSFAH43 38 520399 AP001567 549   1-1859HSFAH43 38 520399 AP001562 550  1-381 HSFAH43 38 520399 AP001562 551 1-663 HSFAH43 38 520399 AP000598 552  1-965 1032-1733 1937-22292257-2448 HSFAH43 38 520399 AL354708 553  1-389 HSFAH43 38 520399AL354708 554  1-606 HSFAH43 38 520399 AL157899 555  1-288 HSFAH43 38520399 AL157762 556  1-366 HSFAH43 38 520399 AC010210 557  1-373 HSFAH4338 520399 AC007849 558  1-89 HSFAH43 38 520399 AC007338 559  1-117HSFAH43 38 520399 AL031601 560  1-417 HSFAH43 38 520399 AC022194 561 1-379 HSFAH43 38 520399 AC022194 562  1-151 HSFAH43 38 520399 AC025624563  1-531 HSFAH43 38 520399 AC025624 564  1-503 HSFAH43 38 520399AC027116 565  1-59  76-532 668-975  995-1151 2499-2635 HSFAH43 38 520399AC026494 566  1-459 HSFAH43 38 520399 AC073118 567  1-553 HSFAH43 38520399 AP000974 568  1-419 HSFAH43 38 520399 AP000445 569  1-331 HSFAH4338 520399 AP000445 570  1-206 HSFAH43 38 520399 AL161645 571  1-410HSFAH43 38 520399 AL161645 572  1-868 HSFAH43 38 520399 AL138682 573 1-543 HSFAH43 38 520399 AL133124 574   1-1512 HSFAH43 38 520399AL133124 575  1-324 HSFAH43 38 520399 AL031726 576   1-3011 3222-3912HSFAH43 38 520399 AC078802 577  1-89 HSFAH43 38 520399 AC073487 578 1-259 HSFAH43 38 520399 AC069525 579  1-179 HSFAH43 38 520399 AC063920580  1-378 HSFAH43 38 520399 AC063920 581  1-386 HSFAH43 38 520399AC021652 582  1-379 HSFAH43 38 520399 AC021652 583  1-151 HSFAH43 38520399 AC018845 584  1-117 HSFAH43 38 520399 AC018840 585  1-433 HSFAH4338 520399 AC016119 586  1-449 HSFAH43 38 520399 AC011432 587  1-523HSFAH43 38 520399 AC011432 588  1-116 HSFAH43 38 520399 AC007623 589 1-167 1804-1865 4649-4745 HSFAH43 38 520399 AC007345 590  1-114 HSFAH4338 520399 AC015521 591   1-1146 HSFAH43 38 520399 AC069153 592  1-327HSFAH43 38 520399 AC010736 593  1-553 HSFAH43 38 520399 AC025802 594 1-503 HSFAH43 38 520399 AC025802 595  1-531 HSFAH43 38 520399 AC018568596  1-239 HSFAH43 38 520399 AC074245 597  1-158 HSFAH43 38 520399AC011823 598  1-120 175-422 779-1165 HSFAH43 38 520399 AC011123 599 1-663 HSFAH43 38 520399 AC011123 600  1-381 HSFAH43 38 520399 AC013708601  1-366 HSFAH43 38 520399 AC037472 602  1-108 HSFAH43 38 520399AL136327 603  1-399 HSPAA60 39 526447 Z98044 604  1-462 HNGBX63 43532615 AL137077 605  1-514 HE2AG50 44 532595 AC026052 606  1-451 830-2549 HE2AG50 44 532595 AL357314 607  1-451  830-2548 HE2AG50 44532595 AC009337 608  1-228  696-1889 HE2AG50 44 532595 AL357314 609 1-84 HCUIN80 45 534128 AC008465 610  1-813 HADCL29 46 532056 AC025153611  1-998 HADCL29 46 532056 AC025153 612  1-134 HADCL29 46 532056AC025153 613  1-88 HAPPS89 47 532135 AC007459 614  1-242 HAPPS89 47532135 AC002369 615  1-586 2559-2651 3329-3426 3756-5088 HAPPS89 47532135 AL390917 616  1-181 HAPPS89 47 532135 AC021401 617  1-319 HAPPS8947 532135 AC013758 618  1-316 HAPPS89 47 532135 AC073909 619  1-286HAPPS89 47 532135 AL354657 620  1-305 HAPPS89 47 532135 AL161634 621 1-300 HAPPS89 47 532135 AC068196 622  1-316 HAPPS89 47 532135 AC021901623  1-283 HAPPS89 47 532135 AC009407 624  1-450 3345-4200 HAPPS89 47532135 AC021921 625  1-170 HAPPS89 47 532135 AC023968 626  1-306 HAPPS8947 532135 AC022051 627  1-294 HAPPS89 47 532135 AC019092 628  1-184HAPPS89 47 532135 AC011233 629  1-258 HAPPS89 47 532135 AC060817 630 1-299 HAPPS89 47 532135 AL353139 631  1-316 HAPPS89 47 532135 AC024337632  1-235 HAPPS89 47 532135 AL358492 633  1-44 HAPPS89 47 532135AC055788 634  1-170 HAPPS89 47 532135 AC023672 635  1-196 HAPPS89 47532135 AC009899 636  1-175 HAPPS89 47 532135 AC009095 637  1-196 HAPPS8947 532135 AP001929 638  1-301 HAPPS89 47 532135 AC012461 639  1-207HAPPS89 47 532135 AC027772 640  1-189 HAPPS89 47 532135 AC027408 641 1-207 HAPPS89 47 532135 AC027584 642  1-162 HAPPS89 47 532135 AC016538643  1-301 HAPPS89 47 532135 AP001992 644  1-301 HAPPS89 47 532135AC067907 645  1-291 HAPPS89 47 532135 AC023309 646  1-193 HAPPS89 47532135 AC041025 647  1-206 3881-3991 HAPPS89 47 532135 AC073446 648 1-140 HAPPS89 47 532135 AC026936 649  1-280 HAPPS89 47 532135 AC024475650  1-187 HAPPS89 47 532135 AC067779 651  1-246 HAPPS89 47 532135AC009858 652  1-39 HAPPS89 47 532135 AC021164 653  1-315 3876-3905HAPPS89 47 532135 AC068682 654  1-153 HAPPS89 47 532135 AC034137 655 1-202 HAPPS89 47 532135 AL356423 656  1-203 HAPPS89 47 532135 AL157775657  1-280 HAPPS89 47 532135 AC068755 658  1-190 HAPPS89 47 532135AC034243 659  1-312 2334-2364 HAPPS89 47 532135 AC021212 660  1-304HAPPS89 47 532135 AC015604 661  1-261 HAPPS89 47 532135 AC036208 662 1-450 3345-4200 HAPPS89 47 532135 AC027234 663  1-318 HAPPS89 47 532135AC068519 664  1-312 HAPPS89 47 532135 AC016319 665  1-191 HAPPS89 47532135 AC011036 666  1-193 HAPPS89 47 532135 AC010853 667  1-195 HAPPS8947 532135 AC055861 668  1-281 HAPPS89 47 532135 AC025869 669  1-323HAPPS89 47 532135 AC073849 670  1-309 HAPPS89 47 532135 AC072051 671 1-310 HAPPS89 47 532135 AC025975 672  1-136 HAPPS89 47 532135 AC015587673  1-273 HAPPS89 47 532135 AL353577 674  1-279 HAPPS89 47 532135AC010073 675  1-243 HAPPS89 47 532135 AL353659 676  1-202 HAPPS89 47532135 AC068289 677  1-192 HAPPS89 47 532135 AC022795 678  1-300 HAPPS8947 532135 AL355975 679  1-322 HAPPS89 47 532135 AC023864 680  1-142HAPPS89 47 532135 AL354696 681  1-181 HAPPS89 47 532135 AC073219 682 1-123 HAPPS89 47 532135 AL139131 683  1-214 HAPPS89 47 532135 AC022766684  1-318 HAPPS89 47 532135 AC027442 685  1-307 HAPPS89 47 532135AC020873 686  1-306 HAPPS89 47 532135 AC002369 687  1-228 HAPPS89 47532135 AL390917 688  1-131 HAPPS89 47 532135 AC068196 689  1-214 HAPPS8947 532135 AC009407 690  1-98 HAPPS89 47 532135 AC019092 691  1-268HAPPS89 47 532135 AC027584 692  1-368 HAPPS89 47 532135 AC073446 693 1-52 2626-2925 HAPPS89 47 532135 AC067779 694   1-1558 1589-4706HAPPS89 47 532135 AC009858 695  1-212 HAPPS89 47 532135 AC036208 696 1-98 HAPPS89 47 532135 AC023864 697   1-1485 1590-4704 HAPPS89 47532135 AC020873 698  1-126 HFGAH44 48 533132 AC023120 699   1-1768HFGAH44 48 533132 AC023120 700  1-127 HFGAH44 48 533132 AC023120 701 1-113 1670-1968 2817-2876 3739-3958 4039-4861 8042-8236  9960-1025310358-10487 11261-11363 12445-12554 17967-18436 19317-19470 19666-1990020264-20363 20475-20779 25579-26082 26905-27377 27555-27983 28656-2899729146-29769 30579-31170 31202-31625 32023-32698 34599-34740 34805-3552035575-37925 HCFBJ91 55 531960 AC022075 702  1-164 479-585 1127-12113462-4318 5484-6362 HCFBJ91 55 531960 AC022075 703  1-157 HHFHP90 56534615 AP001459 704   1-1102 HHFHP90 56 534615 AP000803 705   1-1101HHFHP90 56 534615 AP001362 706   1-1102 HHFHP90 56 534615 AC073481 707 1-34 HHFHP90 56 534615 AC022488 708  1-102 1740-2313 2334-26733403-4504 4670-4849 5046-5195 5301-5477 5601-5841 6181-6671 7025-7084HHFHP90 56 534615 AP001459 709  1-340 HHFHP90 56 534615 AP000803 710 1-340 HHFHP90 56 534615 AP001362 711  1-271 HHFHP90 56 534615 AC022488712  1-549 HHFHP90 56 534615 AC022488 713  1-463 HHLAB07 58 532602AC010764 714  1-750 HFOXE30 59 532120 AL031273 715  1-429 HFOXE30 59532120 AC074234 716  1-397 HFOXE30 59 532120 AL031273 717  1-146 HFOXE3059 532120 AL031273 718  1-175 HBJEL68 60 531924 AL159973 719  1-779HBJEL68 60 531924 AL159973 720  1-217 236-688 HFOXA73 61 850699 AC005866721  1-523 HFOXA73 61 850699 AC007618 722  1-522 HFIUR35 62 532062AC063924 723  1-595 HFIUR35 62 532062 AC063924 724  1-375 HFPDE86 63527728 AC007390 725  1-29 2108-2433 3020-3373 3513-3687 4189-52165348-5697 6109-9606  9821-10357 10998-11303 11348-11630 12256-1268112718-13740 13752-14109 14197-14504 14635-15741 15884-16233 16283-16929HFPDE86 63 527728 AC007390 726  1-429 HFPDE86 63 527728 AC007390 727 1-803

TABLE 1D cDNA Gene No. Clone ID Preferred Indication 1 HSIDU19 Digestive2 HPRSB76 Reproductive 3 HTEIL66 Reproductive 4 HSNAY92 Cancer 5 HSABG21Cancer 6 HSAXB32 Immune/Hematopoetic 7 HPEAD48 Reproductive 8 HPVAB94Reproductive 9 HSAXB81 Immune/Hematopoetic 10 HSAYC21Immune/Hematopoetic 11 HSLCU73 Musculoskeletal 12 HSSFZ70Musculoskeletal 13 HTEIP36 Reproductive 14 HYBAY77 Immune/Hematopoetic,Musculoskeletal 15 HROAE78 Digestive 16 HSAVP17 Immune/Hematopoetic 17HSIEA14 Digestive 18 HSNAQ47 Cancer 19 HODDN65 Reproductive 20 HPEAD79Reproductive 21 HRDED19 Musculoskeletal 22 HSAYS89 Immune/Hematopoetic23 HTODK73 Cancer 24 HSVAM10 Cancer 25 HSNBN57 Cancer 26 HSVBD22 Cancer27 HSAWA27 Immune/Hematopoetic 28 HSFAH43 Cancer 29 HSPAA60 Digestive 30HFAEF57 Neural/Sensory 31 HEGAH43 Digestive, Reproductive 32 HAGDG59Cancer 33 HNGBX63 Immune/Hematopoetic 34 HE2AG50 Digestive, Mixed Fetal,Neural/Sensory 35 HCUIN80 Immune/Hematopoetic 36 HADCL29Connective/Epithelial 37 HAPPS89 Cancer 38 HFGAH44 Cancer 39 HFIHZ96Musculoskeletal 40 HFIUR10 Digestive, Immune/Hematopoetic,Musculoskeletal 41 HLDNA86 Cancer 42 HNGAN75 Immune/Hematopoetic 43HCUIO20 Immune/Hematopoetic 44 HLTEF12 Cancer 45 HCFBJ91Immune/Hematopoetic 46 HHFHP90 Cardiovascular 47 HLYCQ48Immune/Hematopoetic 48 HHLAB07 Digestive, Immune/Hematopoetic 49 HFOXE30Musculoskeletal 50 HBJEL68 Immune/Hematopoetic, Neural/Sensory 51HFOXA73 Musculoskeletal 52 HFIUR35 Musculoskeletal 53 HFPDE86Neural/Sensory

TABLE 1E cDNA Clone AA SEQ ID Gene No. ID NO: Y Biological ActivityExemplary Activity Assay Preferred Indication 2 HPRSB76 68 Production ofIL-10 Assays for production of IL-10 Highly preferred indications andactivation of T- and activation of T-cells are include allergy andasthma. cells. well known in the art and may Additional highly preferredbe used or routinely modified indications include immune to assess theability of and hematopoietic disorders polypeptides of the invention(e.g., as described below under (including antibodies and “ImmuneActivity”, and agonists or antagonists of the “Blood-RelatedDisorders”), invention) to stimulate or autoimmune diseases (e.g.,inhibit production of IL-10 rheumatoid arthritis, systemic and/oractivation of T-cells. lupus erythematosis, Crohn's. Exemplary assaysthat may be disease, multiple sclerosis used or routinely modified toand/or as described below), assess the ability of immunodeficiencies(e.g., as polypeptides and antibodies of described below), boosting a Tthe invention (including cell-mediated immune agonists or antagonists ofthe response, and suppressing a T invention) to modulate IL-10cell-mediated immune production and/or T-cell response. proliferationinclude, for example, assays such as disclosed and/or cited in:Robinson, DS, et al., “Th-2 cytokines in allergic disease” Br Med Bull;56 (4): 956-968 (2000), and Cohn, et al., “T- helper type 2cell-directed therapy for asthma” Phramacology & Therapeutics; 88:187-196 (2000); the contents of each of which are herein incorporated byreference in their entirety. Exemplary cells that may be used accordingto these assays include Th2 cells. IL10 secreted from Th2 cells may bemeasured as a marker of Th2 cell activation. Th2 cells are a class of Tcells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that inducedifferentiation and activation of Th2 cells play a major role in theinitiation and pathogenesis of allergy and asthma. Primary T helper 2cells are generated in in vitro culture under Th2 polarizing conditionsusing peripheral blood lymphocytes isolated from cord blood. 5 HSABG2171 Activation of Assays for the activation of Highly preferredindications transcription transcription through the include asthmaallergy, through STAT-6 Signal Transducers and hypersensitivityreactions, response element in Activators of Transcription inflammation,and immune cells (such (STAT6) response element are inflammatorydisorders. as mast cells). well-known in the art and may Additionalhighly preferred be used or routinely modified indications includeimmune to assess the ability of and hematopoietic disorders polypeptidesof the invention (e.g., as described below under (including antibodiesand “Immune Activity”, and agonists or antagonists of the “Blood-RelatedDisorders”), invention) to regulate STAT6 autoimmune diseases (e.g.,transcription factors and rheumatoid arthritis, systemic modulate theexpression of lupus erythematosis, Crohn's multiple genes. Exemplarydisease, multiple sclerosis assays for transcription and/or as describedbelow), through the STAT6 response immunodeficiencies (e.g., as elementthat may be used or described below), boosting a routinely modified totest mast cell-mediated immune STAT6 response element response, andsuppressing an activity of the polypeptides of mast cell-mediated immunethe invention (including response. antibodies and agonists orantagonists of the invention) include assays disclosed in Berger et al.,Gene 66:1-10 (1998); Cullen and Malm, Methods in Enzymol 216:362- 368(1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988);Georas et al., Blood 92 (12): 4529-4538 (1998); Moffatt et al.,Transplantation 69 (7): 1521-1523 (2000); Curiel et al., Eur J Immunol27 (8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275 (38):29331-29337 (2000); the contents of each of which are hereinincorporated by reference in the entirety. Exemplary cells that may beused according to these assays include mast cells. Mast cells are foundin connective and mucosal tissues throughout the body, and theiractivation via immunoglobulin E-antigen, promoted by T-cell cytokines,is an important component of allergic and asthmatic disease.Furthermore, exemplary assays that may be used or routinely modified toassess the ability of polypeptides of the invention (includingantibodies and agonists or antagonists of the invention) to regulatesignal transduction pathways through STAT-6 transcription factors inmast cells include assays disclosed and/or cited in: Sherman MA., “Therole of STAT6 in mast cell IL-4 production” Immunol Rev; Feb; 179: 48-56(2001); and, Masuda A, et al., “Interleukin- 15 induces rapid tyrosinephosphorylation of STAT6 and the expression of interleukin-4 in mousemast cells” J Biol Chem; Sep 22; 275 (38): 29331-7 (2000); the contentsof each of which are herein incorporated by reference in the entirety. 8HPVAB94 74 Activation of Assays for the activation of Highly preferredindications transcription transcription through the include blooddisorders (e.g., through NFAT Nuclear Factor of Activated T as describedbelow under response element in cells (NFAT) response element “ImmuneActivity”, “Blood- immune cells (such are well-known in the art andRelated Disorders”, and/or as T-cells). may be used or routinely““Cardiovascular Disorders””). modified to assess the ability Highlypreferred indications of polypeptides of the include autoimmune diseasesinvention (including antibodies (e.g., rheumatoid arthritis, andagonists or antagonists of systemic lupus erythematosis, the invention)to regulate multiple sclerosis and/or as NFAT transcription factors anddescribed below), modulate expression of genes immunodeficiencies (e.g.,as involved in described below), boosting a T immunomodulatoryfunctions. cell-mediated immune Exemplary assays for response, andsuppressing a T transcription through the cell-mediated immune NFATresponse element that response. Additional highly may be used orroutinely preferred indications include modified to test NFAT-inflammation and response element activity of inflammatory disorders. Anpolypeptides of the invention additional highly preferred (includingantibodies and indication is infection (e.g., an agonists or antagonistsof the infectious disease as described invention) include assays belowunder “Infectious disclosed in Berger et al., Gene Disease”). Preferred66: 1-10 (1998); Cullen and indications include neoplastic Malm, Methodsin Enzymol diseases (e.g., leukemia, 216: 362-368 (1992); Henthornlymphoma, and/or as described et al., Proc Natl Acad Sci U.S.A. belowunder 85: 6342-6346 (1988); Serfling “Hyperproliferative et al., BiochimBiophys Acta Disorders”). Preferred 1498 (1): 1-18 (2000); De Boerindications include neoplasms et al., Int J Biochem Cell Biol andcancers, such as, for 31 (10): 1221-1236 (1999); example, leukemia,lymphoma, Fraser et al., Eur J Immunol and prostate, breast, lung, 29(3): 838-844 (1999); and colon, pancreatic, esophageal, Yeseen et al., JBiol Chem stomach, brain, liver and 268 (19): 14285-14293 (1993),urinary cancer. Other preferred the contents of each of whichindications include benign are herein incorporated by dysproliferativedisorders and reference in its entirety. T pre-neoplastic conditions,such cells that may be used as, for example, hyperplasia, according tothese assays are metaplasia, and/or dysplasia. publicly available (e.g.,Preferred indications also through the ATCC). include anemia,pancytopenia, Exemplary human T cells that leukopenia, thrombocytopenia,may be used according to these Hodgkin's disease, acute assays includethe SUPT cell lymphocytic anemia (ALL), line, which is a suspensionplasmacytomas, multiple culture of IL-2 and IL-4 myeloma, Burkitt'slymphoma, responsive T cells. arthritis, AIDS, granulomatous disease,inflammatory bowel disease, sepsis, neutropenia, neutrophilia,psoriasis, suppression of immune reactions to transplanted organs andtissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis,meningitis, Lyme Disease, asthma and allergy. 13 HTEIP36 79 Activationof Assays for the activation of Highly preferred indicationstranscription transcription through the include inflammation and throughNFKB NFKB response element are inflammatory disorders. response elementin well-known in the art and may Highly preferred indications immunecells (such be used or routinely modified include blood disorders (e.g.,as T-cells). to assess the ability of as described below underpolypeptides of the invention “Immune Activity”, “Blood- (includingantibodies and Related Disorders”, and/or agonists or antagonists of the““Cardiovascular Disorders””). invention) to regulate NFKB Highlypreferred indications transcription factors and include autoimmunediseases modulate expression of (e.g., rheumatoid arthritis,immunomodulatory genes. systemic lupus erythematosis, Exemplary assaysfor multiple sclerosis and/or as transcription through the describedbelow), and NFKB response element that immunodeficiencies (e.g., as maybe used or routinely described below). An modified to test NFKB-additional highly preferred response element activity of indication isinfection (e.g., polypeptides of the invention AIDS, and/or aninfectious (including antibodies and disease as described below agonistsor antagonists of the under “Infectious Disease”). invention) includeassays Highly preferred indications disclosed in Berger et al., Geneinclude neoplastic diseases 66: 1-10 (1998); Cullen and (e.g., melanoma,leukemia, Malm, Methods in Enzymol lymphoma, and/or as described 216:362-368 (1992); Henthorn below under et al., Proc Natl Acad Sci U.S.A.“Hyperproliferative 85: 6342-6346 (1988); Black et Disorders”). Highlypreferred al., Virus Gnes 15 (2): 105-117 indications include neoplasms(1997); and Fraser et al., and cancers, such as, for 29 (3): 838-844(1999), the example, melanoma, renal cell contents of each of which arecarcinoma, leukemia, herein incorporated by lymphoma, and prostate,reference in its entirety. breast, lung, colon, pancreatic, Exemplaryhuman T cells, esophageal, stomach, brain, such as the MOLT4, that mayliver and urinary cancer. Other be used according to these preferredindications include assays are publicly available benigndysproliferative (e.g., through the ATCC). disorders and pre-neoplasticconditions, such as, for example, hyperplasia, metaplasia, and/ordysplasia. Preferred indications also include anemia, pancytopenia,leukopenia, thrombocytopenia, Hodgkin's disease, acute lymphocyticanemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma,arthritis, AIDS, granulomatous disease, inflammatory bowel disease,sepsis, neutropenia, neutrophilia, psoriasis, hemophilia,hypercoagulation, diabetes mellitus, endocarditis, meningitis, LymeDisease, suppression of immune reactions to transplanted organs, asthmaand allergy. 13 HTEIP36 79 Production of IL-10 Assays for production ofIL-10 Highly preferred indications and activation of T- and activationof T-cells are include allergy and asthma. cells. well known in the artand may Additional highly preferred be used or routinely modifiedindications include immune to assess the ability of and hematopoieticdisorders polypeptides of the invention (e.g., as described below under(including antibodies and “Immune Activity”, and agonists or antagonistsof the “Blood-Related Disorders”), invention) to stimulate or autoimmunediseases (e.g., inhibit production of IL-10 rheumatoid arthritis,systemic and/or activation of T-cells. lupus erythematosis, Crohn'sExemplary assays that may be disease, multiple sclerosis used orroutinely modified to and/or as described below), assess the ability ofimmunodeficiencies (e.g., as polypeptides and antibodies of describedbelow), boosting a T the invention (including cell-mediated immuneagonists or antagonists of the response, and suppressing a T invention)to modulate IL-10 cell-mediated immune production and/or T-cellresponse. proliferation include, for example, assays such as disclosedand/or cited in: Robinson, DS, et al., “Th-2 cytokines in allergicdisease” Br Med Bull; 56 (4): 956-968 (2000), and Cohn, et al., “T-helper type 2 cell-directed therapy for asthma” Phramacology &Therapeutics; 88: 187-196 (2000); the contents of each of which areherein incorporated by reference in their entirety. Exemplary cells thatmay be used according to these assays include Th2 cells. IL10 secretedfrom Th2 cells may be measured as a marker of Th2 cell activation. Th2cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6.Factors that induce differentiation and activation of Th2 cells play amajor role in the initiation and pathogenesis of allergy and asthma.Primary T helper 2 cells are generated in in vitro culture under Th2polarizing conditions using peripheral blood lymphocytes isolated fromcord blood. 19 HODDN65 85 Production of Assays for measuring Preferredembodiments of the ICAM-1 expression of ICAM-1 are invention includeusing well-known in the art and may polypeptides of the invention beused or routinely modified (or antibodies, agonists, or to assess theability of antagonists thereof) in polypeptides of the inventiondetection, diagnosis, (including antibodies and prevention, and/ortreatment of agonists or antagonists of the Inflammation, Vascularinvention) to regulate ICAM-1 Disease, Athereosclerosis, expression.Exemplary assays Restenosis, and Stroke that may be used or routinelymodified to measure ICAM-1 expression include assays disclosed in:Takacs P, et al, FASEB J, 15 (2): 279-281 (2001); and, Miyamoto K, etal., Am J Pathol, 156 (5): 1733-1739 (2000), the contents of each ofwhich is herein incorporated by reference in its entirety. Cells thatmay be used according to these assays are publicly available (e.g.,through the ATCC) and/or may be routinely generated. Exemplary cellsthat may be used according to these assays include microvascularendothelial cells (MVEC). 20 HPEAD79 86 Regulation of Assays for theregulation of A highly preferred transcription via transcription throughthe indication is diabetes mellitus. DMEF1 response DMEF1 responseelement are An additional highly preferred element in well-known in theart and may indication is a complication adipocytes and pre- be used orroutinely modified associated with diabetes (e.g., adipocytes to assessthe ability of diabetic retinopathy, diabetic polypeptides of theinvention nephropathy, kidney disease (including antibodies and (e.g.,renal failure, agonists or antagonists of the nephropathy and/or otherinvention) to activate the diseases and disorders as DMEF1 responseelement in a described in the ““Renal reporter construct (such as thatDisorders”” section below), containing the GLUT4 diabetic neuropathy,nerve promoter) and to regulate disease and nerve damage insulinproduction. The (e.g., due to diabetic DMEF1 response element isneuropathy), blood vessel present in the GLUT4 blockage, heart disease,stroke, promoter and binds to MEF2 impotence (e.g., due to diabetictranscription factor and another neuropathy or blood vesseltranscription factor that is blockage), seizures, mental required forinsulin regulation confusion, drowsiness, of Glut4 expression inskeletal nonketotic hyperglycemic- muscle. GLUT4 is the primaryhyperosmolar coma, insulin-responsive glucose cardiovascular disease(e.g., transporter in fat and muscle heart disease, atherosclerosis,tissue. Exemplary assays that microvascular disease, may be used orroutinely hypertension, stroke, and other modified to test for DMEF1diseases and disorders as response element activity (in described in theadipocytes and pre-adipocytes) ““Cardiovascular Disorders”” bypolypeptides of the section below), dyslipidemia, invention (includingantibodies endocrine disorders (as and agonists or antagonists ofdescribed in the ““Endocrine the invention) include assays Disorders””section below), disclosed in Thai, M.V., et al., J neuropathy, visionimpairment Biol Chem, 273 (23): 14285-92 (e.g., diabetic retinopathy and(1998); Mora, S., et al., J Biol blindness), ulcers and impaired Chem,275 (21): 16323-8 wound healing, and infection (2000); Liu, M.L., etal., J Biol (e.g., infectious diseases and Chem, 269 (45): 28514-21disorders as described in the (1994); ““Identification of a ““InfectiousDiseases”” section 30-base pair regulatory below, especially of theelement and novel DNA urinary tract and skin), carpal binding proteinthat regulates tunnel syndrome and the human GLUT4 promoter inDupuytren's contracture). transgenic mice””, J Biol An additional highlypreferred Chem. 2000 Aug indication is obesity and/or 4; 275 (31):23666-73; Berger, et complications associated with al., Gene 66: 1-10(1988); and, obesity. Additional highly Cullen, B., et al., Methods inpreferred indications include Enzymol. 216: 362-368 (1992), weight lossor alternatively, the contents of each of which weight gain. Additionalis herein incorporated by highly preferred indications are reference inits entirety. complications associated with Adipocytes andpre-adipocytes insulin resistance. that may be used according to theseassays are publicly available (e.g., through the ATCC) and/or may beroutinely generated. Exemplary cells that may be used according to theseassays include the mouse 3T3-L1 cell line which is an adherent mousepreadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of3T3 fibroblasts developed through clonal isolation. These cells undergoa pre-adipocyte to adipose-like conversion under appropriatedifferentiation culture conditions. 23 HTODK73 89 Activation of Assaysfor the activation of Highly preferred indications transcriptiontranscription through the include blood disorders (e.g., through NFATNuclear Factor of Activated T as described below under response inimmune cells (NFAT) response element “Immune Activity”, “Blood- cells(such as T- are well-known in the art and Related Disorders”, and/orcells). may be used or routinely ““Cardiovascular Disorders””). modifiedto assess the ability Highly preferred indications of polypeptides ofthe include autoimmune diseases invention (including antibodies (e.g.,rheumatoid arthritis, and agonists or antagonists of systemic lupuserythematosis, the invention) to regulate multiple sclerosis and/or asNFAT transcription factors and described below), modulate expression ofgenes immunodeficiencies (e.g., as involved in described below),boosting a T immunomodulatory functions. cell-mediated immune Exemplaryassays for response, and suppressing a T transcription through thecell-mediated immune NFAT response element that response. Additionalhighly may be used or routinely preferred indications include modifiedto test NFAT- inflammation and response element activity of inflammatorydisorders. An polypeptides of the invention additional highly preferred(including antibodies and indication is infection (e.g., an agonists orantagonists of the infectious disease as described invention) includeassays below under “Infectious disclosed in Berger et al., GeneDisease”). Preferred 66: 1-10 (1998); Cullen and indications includeneoplastic Malm, Methods in Enzymol diseases (e.g., leukemia, 216:362-368 (1992); Henthorn lymphoma, and/or as described et al., Proc NatlAcad Sci U.S.A. below under 85: 6342-6346 (1988); Serfling“Hyperproliferative et al., Biochim Biophys Acta Disorders”). Preferred1498 (1): 1-18 (2000); De Boer indications include neoplasms et al., IntJ Biochem Cell Biol and cancers, such as, for 31 (10): 1221-1236 (1999);example, leukemia, lymphoma, Fraser et al., Eur J Immunol and prostate,breast, lung, 29 (3): 838-844 (1999); and colon, pancreatic, esophageal,Yeseen et al., J Biol Chem stomach, brain, liver and 268 (19):14285-14293 (1993), urinary cancer. Other preferred the contents of eachof which indications include benign are herein incorporated bydysproliferative disorders and reference in its entirety. Tpre-neoplastic conditions, such cells that may be used as, for example,hyperplasia, according to these assays are metaplasia, and/or dysplasia.publicly available (e.g., Preferred indications also through the ATCC).include anemia, pancytopenia, Exemplary human T cells that leukopenia,thrombocytopenia, may be used according to these Hodgkin's disease,acute assays include the JURKAT lymphocytic anemia (ALL), cell line,which is a suspension plasmacytomas, multiple culture of leukemia cellsthat myeloma, Burkitt's lymphoma, produce IL-2 when stimulated.arthritis, AIDS, granulomatous disease, inflammatory bowel disease,sepsis, neutropenia, neutrophilia, psoriasis, suppression of immunereactions to transplanted organs and tissues, hemophilia,hypercoagulation, diabetes mellitus, endocarditis, meningitis, LymeDisease, asthma and allergy. 24 HSVAM10 90 Production of IFNgamma FMAT.IFNg plays A highly preferred IFNgamma using a a central role in theimmune embodiment of the invention T cells system and is considered tobe includes a method for a proinflammatory cytokine. stimulating theproduction of IFNg promotes TH1 and IFNg. An alternative highly inhibitsTH2 differentiation; preferred embodiment of the promotes IgG2a andinhibits invention includes a method IgE secretion; induces forinhibiting the production of macrophage activation; and IFNg. Highlypreferred increases MHC expression. indications include blood Assays forimmunomodulatory disorders (e.g., as described proteins produced by Tcells below under “Immune and NK cells that regulate a Activity”,“Blood-Related variety of inflammatory Disorders”, and/or activities andinhibit TH2 ““Cardiovascular Disorders””), helper cell functions arewell and infection (e.g., viral known in the art and may be infections,tuberculosis, used or routinely modified to infections associated withassess the ability of chronic granulomatosus polypeptides of theinvention disease and malignant (including antibodies and osteoporosis,and/or as agonists or antagonists of the described below underinvention) to mediate “Infectious Disease”). Highly immunomodulation,regulate preferred indications include inflammatory activities,autoimmune disease (e.g., modulate TH2 helper cell rheumatoid arthritis,systemic function, and/or mediate lupus erythematosis, multiple humoralor cell-mediated sclerosis and/or as described immunity. Exemplaryassays below), immunodeficiency that test for (e.g., as describedbelow), immunomodulatory proteins boosting a T cell-mediated evaluatethe production of immune response, and cytokines, such as Interferonsuppressing a T cell-mediated gamma (IFNg), and the immune response.Additional activation of T cells. Such highly preferred indicationsassays that may be used or include inflammation and routinely modifiedto test inflammatory disorders. immunomodulatory activity of Additionalpreferred polypeptides of the invention indications include idiopathic(including antibodies and pulmonary fibrosis. Highly agonists orantagonists of the preferred indications include invention) include theassays neoplastic diseases (e.g., disclosed in Miraglia et al., Jleukemia, lymphoma, Biomolecular Screening 4: 193-204 melanoma, and/oras described (1999); Rowland et al., below under ““Lymphocytes: apractical “Hyperproliferative approach”” Chapter 6: 138-160 Disorders”).Highly preferred (2000); Gonzalez et al., J Clin indications includeneoplasms Lab Anal 8 (5): 225-233 (1995); and cancers, such as, forBilliau et al., Ann NY Acad example, leukemia, lymphoma, Sci 856: 22-32(1998); Boehm melanoma, and prostate, et al., Annu Rev Immunol breast,lung, colon, pancreatic, 15: 749-795 (1997), and esophageal, stomach,brain, Rheumatology (Oxford) liver and urinary cancer. Other 38 (3):214-20 (1999), the preferred indications include contents of each ofwhich are benign dysproliferative herein incorporated by disorders andpre-neoplastic reference in its entirety. conditions, such as, for HumanT cells that may be example, hyperplasia, used according to these assaysmetaplasia, and/or dysplasia. may be isolated using Preferredindications include techniques disclosed herein or anemia, pancytopenia,otherwise known in the art. leukopenia, thrombocytopenia, Human T cellsare primary Hodgkin's disease, acute human lymphocytes that lymphocyticanemia (ALL), mature in the thymus and plasmacytomas, multiple express aT Cell receptor and myeloma, Burkitt's lymphoma, CD3, CD4, or CD8. Thesearthritis, AIDS, granulomatous cells mediate humoral or cell- disease,inflammatory bowel mediated immunity and may disease, sepsis,neutropenia, be preactivated to enhance neutrophilia, psoriasis,responsiveness to suppression of immune immunomodulatory factors.reactions to transplanted organs and tissues, hemophilia,hypercoagulation, diabetes mellitus, endocarditis, meningitis, LymeDisease, asthma and allergy. 30 HFAEF57 96 Regulation of Assays for theregulation of A highly preferred transcription transcription through theFAS indication is diabetes mellitus. through the FAS promoter elementare well- An additional highly preferred promoter element known in theart and may be indication is a complication in hepatocytes used orroutinely modified to associated with diabetes (e.g., assess the abilityof diabetic retinopathy, diabetic polypeptides of the inventionnephropathy, kidney disease (including antibodies and (e.g., renalfailure, agonists or antagonists of the nephropathy and/or otherinvention) to activate the FAS diseases and disorders as promoterelement in a reporter described in the ““Renal construct and to regulateDisorders”” section below), transcription of FAS, a key diabeticneuropathy, nerve enzyme for lipogenesis. FAS disease and nerve damagepromoter is regulated by many (e.g., due to diabetic transcriptionfactors including neuropathy), blood vessel SREBP. Insulin increases FASblockage, heart disease, stroke, gene transcription in livers ofimpotence (e.g., due to diabetic diabetic mice. This neuropathy or bloodvessel stimulation of transcription is blockage), seizures, mental alsosomewhat glucose confusion, drowsiness, dependent. Exemplary assaysnonketotic hyperglycemic- that may be used or routinely hyperosmolarcoma, modified to test for FAS cardiovascular disease (e.g., promoterelement activity (in heart disease, atherosclerosis, hepatocytes) bypolypeptides microvascular disease, of the invention (includinghypertension, stroke, and other antibodies and agonists or diseases anddisorders as antagonists of the invention) described in the includeassays disclosed in ““Cardiovascular Disorders”” Xiong, S., et al., ProcNatl section below), dyslipidemia, Acad Sci U.S.A., 97 (8): 3948-53endocrine disorders (as (2000); Roder, K., et al., described in the““Endocrine Eur J Biochem, 260 (3): 743-51 Disorders”” section below),(1999); Oskouian B, et al., neuropathy, vision impairment Biochem J, 317(Pt 1): 257-65 (e.g., diabetic retinopathy and (1996); Berger, et al.,Gene blindness), ulcers and impaired 66: 1-10 (1988); and, Cullen, woundhealing, and infection B., et al., Methods in Enzymol. (e.g., infectiousdiseases and 216: 362-368 (1992), the disorders as described in thecontents of each of which is ““Infectious Diseases”” section hereinincorporated by below, especially of the reference in its entirety.urinary tract and skin), carpal Hepatocytes that may be used tunnelsyndrome and according to these assays, such Dupuytren's contracture).as H4IIE cells, are publicly An additional highly preferred available(e.g., through the indication is obesity and/or ATCC) and/or may becomplications associated with routinely generated. obesity. Additionalhighly Exemplary hepatocytes that preferred indications include may beused according to these weight loss or alternatively, assays include ratliver weight gain. Additional hepatoma cell line(s) inducible highlypreferred indications are with glucocorticoids, insulin, complicationsassociated with or cAMP derivatives. insulin resistance. 31 HEGAH43 97Endothelial Cell Caspase Apoptosis. Assays for A highly preferredApoptosis caspase apoptosis are well embodiment of the invention knownin the art and may be includes a method for used or routinely modifiedto stimulating endothelial cell assess the ability of growth. Analternative highly polypeptides of the invention preferred embodiment ofthe (including antibodies and invention includes a method agonists orantagonists of the for inhibiting endothelial cell invention) to promotecaspase growth. A highly preferred protease-mediated apoptosis.embodiment of the invention Induction of apoptosis in includes a methodfor endothelial cells supporting the stimulating endothelial cellvasculature of tumors is proliferation. An alternative associated withtumor highly preferred embodiment regression due to loss of tumor of theinvention includes a blood supply. Exemplary method for inhibitingassays for caspase apoptosis endothelial cell proliferation. that may beused or routinely A highly preferred modified to test capase embodimentof the invention apoptosis activity of includes a method forpolypeptides of the invention stimulating apoptosis of (includingantibodies and endothelial cells. An agonists or antagonists of thealternative highly preferred invention) include the assays embodiment ofthe invention disclosed in Lee et al., FEBS includes a method for Lett485 (2-3): 122-126 (2000); inhibiting (e.g., decreasing) Nor et al., JVasc Res 37 (3): apoptosis of endothelial cells. 209-218 (2000); andKarsan A highly preferred and Harlan, J Atheroscler embodiment of theinvention Thromb 3 (2): 75-80 (1996); includes a method for the contentsof each of which stimulating angiogenisis. An are herein incorporated byalternative highly preferred reference in its entirety. embodiment ofthe invention Endothelial cells that may be includes a method for usedaccording to these assays inhibiting angiogenesis. A are publiclyavailable (e.g., highly preferred embodiment through commercialsources). of the invention includes a Exemplary endothelial cells methodfor reducing cardiac that may be used according to hypertrophy. Analternative these assays include bovine highly preferred embodimentaortic endothelial cells of the invention includes a (bAEC), which arean example method for inducing cardiac of endothelial cells which linehypertrophy. Highly blood vessels and are involved preferred indicationsinclude in functions that include, but neoplastic diseases (e.g., as arenot limited to, described below under angiogenesis, vascular“Hyperproliferative permeability, vascular tone, Disorders”), anddisorders of and immune cell extravasation. the cardiovascular system(e.g., heart disease, congestive heart failure, hypertension, aorticstenosis, cardiomyopathy, valvular regurgitation, left ventriculardysfunction, atherosclerosis and atherosclerotic vascular disease,diabetic nephropathy, intracardiac shunt, cardiac hypertrophy,myocardial infarction, chronic hemodynamic overload, and/or as describedbelow under “Cardiovascular Disorders”). Highly preferred indicationsinclude cardiovascular, endothelial and/or angiogenic disorders (e.g.,systemic disorders that affect vessels such as diabetes mellitus, aswell as diseases of the vessels themselves, such as of the arteries,capillaries, veins and/or lymphatics). Highly preferred are indicationsthat stimulate angiogenesis and/or cardiovascularization. Highlypreferred are indications that inhibit angiogenesis and/orcardiovascularization. Highly preferred indications includeantiangiogenic activity to treat solid tumors, leukemias, and Kaposi'ssarcoma, and retinal disorders. Highly preferred indications includeneoplasms and cancer, such as, Kaposi's sarcoma, hemangioma (capillaryand cavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, lymphangioma,lymphangiosarcoma. Highly preferred indications also include cancerssuch as, prostate, breast, lung, colon, pancreatic, esophageal, stomach,brain, liver, and urinary cancer. Preferred indications include benigndysproliferative disorders and pre-neoplastic conditions, such as, forexample, hyperplasia, metaplasia, and/or dysplasia. Highly preferredindications also include arterial disease, such as, atherosclerosis,hypertension, coronary artery disease, inflammatory vasculitides,Reynaud's disease and Reynaud's phenomenom, aneurysms, restenosis;venous and lymphatic disorders such as thrombophlebitis, lymphangitis,and lymphedema; and other vascular disorders such as peripheral vasculardisease, and cancer. Highly preferred indications also include traumasuch as wounds, burns, and injured tissue (e.g., vascular injury suchas, injury resulting from balloon angioplasty, and atheroschleroticlesions), implant fixation, scarring, ischemia reperfusion injury,rheumatoid arthritis, cerebrovascular disease, renal diseases such asacute renal failure, and osteoporosis. Additional highly preferredindications include stroke, graft rejection, diabetic or otherretinopathies, thrombotic and coagulative disorders, vascularitis, lymphangiogenesis, sexual disorders, age-related macular degeneration, andtreatment/ prevention of endometriosis and related conditions.Additional highly preferred indications include fibromas, heart disease,cardiac arrest, heart valve disease, and vascular disease. Preferredindications include blood disorders (e.g., as described below under“Immune Activity”, “Blood- Related Disorders”, and/or ““CardiovascularDisorders””). Preferred indications include autoimmune diseases (e.g.,rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosisand/or as described below) and immunodeficiencies (e.g., as describedbelow). Additional preferred indications include inflammation andinflammatory disorders (such as acute and chronic inflammatory diseases,e.g., inflammatory bowel disease and Crohn's disease), and painmanagement. 32 HAGDG59 98 Production of MIP1 MIP-1alpha FMAT. Assays Ahighly preferred alpha for immunomodulatory embodiment of the inventionproteins produced by activated includes a method for dendritic cellsthat upregulate stimulating MIP1a production. monocyte/macrophage and TAn alternative highly preferred cell chemotaxis are well embodiment ofthe invention known in the art and may be includes a method for used orroutinely modified to inhibiting (e.g., reducing) assess the ability ofMIP1a production. polypeptides of the invention A highly preferred(including antibodies and indication is infection (e.g., an agonists orantagonists of the infectious disease as described invention) to mediatebelow under “Infectious immunomodulation, modulate Disease”).chemotaxis, and modulate T Preferred indications cell differentiation.Exemplary include blood disorders (e.g., assays that test for asdescribed below under immunomodulatory proteins “Immune Activity”,“Blood- evaluate the production of Related Disorders”, and/orchemokines, such as “Cardiovascular Disorders”). macrophage inflammatoryHighly preferred indications protein 1 alpha (MIP-1a), and includeautoimmune diseases the activation of (e.g., rheumatoid arthritis,monocytes/macrophages and T systemic lupus erythematosis, cells. Suchassays that may be multiple sclerosis and/or as used or routinelymodified to described below) and test immunomodulatory andimmunodeficiencies (e.g., as chemotaxis activity of described below).Additional polypeptides of the invention highly preferred indications(including antibodies and include inflammation and agonists orantagonists of the inflammatory disorders. invention) include assaysPreferred indications also disclosed in Miraglia et al., J includeanemia, pancytopenia, Biomolecular Screening 4: 193-204 leukopenia,thrombocytopenia, (1999); Rowland et al., Hodgkin's disease, acute“Lymphocytes: a practical lymphocytic anemia (ALL), approach” Chapter 6:138-160 plasmacytomas, multiple (2000); Satthaporn and myeloma,Burkitt's lymphoma Eremin, J R Coll Surg Ednb arthritis, AIDS,granulomatous 45 (1): 9-19 (2001); Drakes et al., disease, inflammatorybowel Transp Immunol 8 (1): 17-29 disease, sepsis, neutropenia, (2000);Verhasselt et al., J neutrophilia, psoriasis, Immunol 158: 2919-2925suppression of immune (1997); and Nardelli et al., J reactions totransplanted Leukoc Biol 65: 822-828 organs and tissues, hemophilia(1999), the contents of each of hypercoagulation, diabetes which areherein incorporated mellitus, endocarditis, by reference in itsentirety. meningitis, Lyme Disease, Human dendritic cells that mayasthma, and allergy. be used according to these Preferred indicationsalso assays may be isolated using include neoplastic diseases techniquesdisclosed herein or (e.g., leukemia, lymphoma, otherwise known in theart. and/or as described below Human dendritic cells are under“Hyperproliferative antigen presenting cells in Disorders”). Highlypreferred suspension culture, which, indications include neoplasms whenactivated by antigen and cancers, such as, leukemia, and/or cytokines,initiate and lymphoma, prostate, breast, upregulate T cell proliferationlung, colon, pancreatic, and functional activities. esophageal, stomach,brain, liver, and urinary cancer. Other preferred indications includebenign dysproliferative disorders and pre-neoplastic conditions, suchas, for example, hyperplasia, metaplasia, and/or dysplasia. 32 HAGDG5998 T cell proliferation T Cell Proliferation. Assays A highly preferredfor T cell proliferation, embodiment of the invention measured bytridiated (3H) includes a method for Thymidine incorporation, arestimulating T cell proliferation. well known in the art and may Analternative highly preferred be used or routinely modified embodiment ofthe invention to assess the ability of the includes a method forpolypeptides of the invention inhibiting T cell proliferation.(including antibodies and Preferred indications include agonists orantagonists of the blood disorders (e.g., as invention) to mediatedescribed below under immunomodulation, modulate “Immune Activity”,“Blood- T cell proliferation and Related Disorders”, and/or mediatehumoral and/or cell- “Cardiovascular Disorders”), mediated immunity. andinfection (e.g., an Exemplary assays that may be infectious disease asdescribed used or routinely modified to below under “Infectious assessthe ability of Disease”). Highly preferred polypeptides of the inventionindications include (including antibodies and autoimmune diseases (e.g.,agonists or antagonists of the rheumatoid arthritis, systemic invention)to modulate T cell lupus erythematosis, multiple proliferation, asmeasured by sclerosis and/or as described 3H Thymidine incorporation,below), immunodeficiencies include the assays disclosed in (e.g., asdescribed below), Rowland et al., “Lymphocytes: boosting a Tcell-mediated a practical approach” Chapter immune response, and 6:138-160 (2000); Gansbacher suppressing a T cell-mediated and Zier, CellImmunol immune response. Additional 117 (1): 22-34 (1988); and highlypreferred indications Werfel et al., Allergy include inflammation and 52(4): 465-469 (1997), the inflammatory disorders. contents of each ofwhich are Highly preferred indications herein incorporated by includeneoplastic diseases reference in its entirety. (e.g., leukemia,lymphoma, Human T cells that may be and/or as described below usedaccording to these assays under “Hyperproliferative may be isolatedusing Disorders”). Highly preferred techniques disclosed herein orindications include neoplasms otherwise known in the art. and cancers,such as, for Human T cells are primary example, leukemia, lymphoma humanlymphocytes that (e.g., T cell lymphoma, mature in the thymus andBurkitt's lymphoma, non- express a T cell receptor and Hodgkinslymphoma, CD3, CD4, or CD8. These Hodgkin's disease), melanoma, cellsmediate humoral or cell- and prostate, breast, lung, mediated immunityand may colon, pancreatic, esophageal, be preactivated to enhancestomach, brain, liver and responsiveness to urinary cancer. Otherpreferred immunomodulatory factors. indications include benigndysproliferative disorders and pre-neoplastic conditions, such as, forexample, hyperplasia, metaplasia, and/or dysplasia. Preferredindications include anemia, pancytopenia, leukopenia, thrombocytopenia,acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma,arthritis, AIDS, granulomatous disease, inflammatory bowel disease,sepsis, neutropenia, neutrophilia, psoriasis, suppression of immunereactions to transplanted organs and tissues, hemophilia,hypercoagulation, diabetes mellitus, endocarditis, meningitis, LymeDisease, and asthma and allergy. 32 HAGDG59 98 T cell proliferation TCell Proliferation. Assays A highly preferred for T cell proliferation,embodiment of the invention measured by tridiated (3H) includes a methodfor Thymidine incorporation, are stimulating T cell proliferation. wellknown in the art and may An alternative highly preferred be used orroutinely modified embodiment of the invention to assess the ability ofthe includes a method for polypeptides of the invention inhibiting Tcell proliferation. (including antibodies and Preferred indicationsinclude agonists or antagonists of the blood disorders (e.g., asinvention) to mediate described below under immunomodulation, modulate“Immune Activity”, “Blood- T cell proliferation and Related Disorders”,and/or mediate humoral and/or cell- “Cardiovascular Disorders”),mediated immunity. and infection (e.g., an Exemplary assays that may beinfectious disease as described used or routinely modified to belowunder “Infectious assess the ability of Disease”). Highly preferredpolypeptides of the invention indications include (including antibodiesand autoimmune diseases (e.g., agonists or antagonists of the rheumatoidarthritis, systemic invention) to modulate T cell lupus erythematosis,multiple proliferation, as measured by sclerosis and/or as described 3HThymidine incorporation, below), immunodeficiencies include the assaysdisclosed in (e.g., as described below), Rowland et al., “Lymphocytes:boosting a T cell-mediated a practical approach” Chapter immuneresponse, and 6: 138-160 (2000); Gansbacher suppressing a Tcell-mediated and Zier, Cell Immunol immune response. Additional 117(1): 22-34 (1988); and highly preferred indications Werfel et al.,Allergy include inflammation and 52 (4): 465-469 (1997), theinflammatory disorders. contents of each of which are Highly preferredindications herein incorporated by include neoplastic diseases referencein its entirety. (e.g., leukemia, lymphoma, Human T cells that may beand/or as described below used according to these assays under“Hyperproliferative may be isolated using Disorders”). Highly preferredtechniques disclosed herein or indications include neoplasms otherwiseknown in the art. and cancers, such as, for Human T cells are primaryexample, leukemia, lymphoma human lymphocytes that (e.g., T celllymphoma, mature in the thymus and Burkitt's lymphoma, non- express a Tcell receptor and Hodgkins lymphoma, CD3, CD4, or CD8. These Hodgkin'sdisease), melanoma, cells mediate humoral or cell- and prostate, breast,lung, mediated immunity and may colon, pancreatic, esophageal, bepreactivated to enhance stomach, brain, liver and responsiveness tourinary cancer. Other preferred immunomodulatory factors. indicationsinclude benign dysproliferative disorders and pre-neoplastic conditions,such as, for example, hyperplasia, metaplasia, and/or dysplasia.Preferred indications include anemia, pancytopenia, leukopenia,thrombocytopenia, acute lymphocytic anemia (ALL), plasmacytomas,multiple myeloma, arthritis, AIDS, granulomatous disease, inflammatorybowel disease, sepsis, neutropenia, neutrophilia, psoriasis, suppressionof immune reactions to transplanted organs and tissues, hemophilia,hypercoagulation, diabetes mellitus, endocarditis, meningitis, LymeDisease, and asthma and allergy. 40 HFIUR10 106 Regulation of Assays forthe regulation of A highly preferred viability and viability andproliferation of indication is diabetes mellitus. proliferation of cellsin vitro are well-known in An additional highly preferred pancreaticbeta the art and may be used or indication is a complication cells.routinely modified to assess associated with diabetes (e.g., the abilityof polypeptides of diabetic retinopathy, diabetic the invention(including nephropathy, kidney disease antibodies and agonists or (e.g.,renal failure, antagonists of the invention) to nephropathy and/or otherregulate viability and diseases and disorders as proliferation ofpancreatic beta described in the ““Renal cells. For example, the CellDisorders”” section below), Titer-Glo luminescent cell diabeticneuropathy, nerve viability assay measures the disease and nerve damagenumber of viable cells in (e.g., due to diabetic culture based onquantitation neuropathy), blood vessel of the ATP present whichblockage, heart disease, stroke, signals the presence of impotence(e.g., due to diabetic metabolically active cells. neuropathy or bloodvessel Exemplary assays that may be blockage), seizures, mental used orroutinely modified to confusion, drowsiness, test regulation ofviability and nonketotic hyperglycemic- proliferation of pancreatic betahyperosmolar coma, cells by polypeptides of the cardiovascular disease(e.g., invention (including antibodies heart disease, atherosclerosis,and agonists or antagonists of microvascular disease, the invention)include assays hypertension, stroke, and other disclosed in: Ohtani KI,et al., diseases and disorders as Endocrinology, 139 (1): 172-8described in the (1998); Krautheim A, et al, ““CardiovascularDisorders”” Exp Clin Endocrinol Diabetes, section below), dyslipidemia,107 (1): 29-34 (1999), the endocrine disorders (as contents of each ofwhich is described in the ““Endocrine herein incorporated by Disorders””section below), reference in its entirety. neuropathy, vision impairmentPancreatic cells that may be (e.g., diabetic retinopathy and usedaccording to these assays blindness), ulcers and impaired are publiclyavailable (e.g., wound healing, and infection through the ATCC) and/or(e.g., infectious diseases and may be routinely generated. disorders asdescribed in the Exemplary pancreatic cells that ““Infectious Diseases””section may be used according to these below, especially of the assaysinclude HITT15 Cells. urinary tract and skin), carpal HITT15 are anadherent tunnel syndrome and epithelial cell line establishedDupuytren's contracture). from Syrian hamster islet cells An additionalhighly preferred transformed with SV40. These indication is obesityand/or cells express glucagon, complications associated withsomatostatin, and obesity. Additional highly glucocorticoid receptors.The preferred indications include cells secrete insulin, which is weightloss or alternatively, stimulated by glucose and weight gain. glucagonand suppressed by Additional highly preferred somatostatin orindications are complications glucocorticoids. ATTC# CRL- associatedwith insulin 1777 Refs: Lord and resistance. Ashcroft. Biochem. J. 219:547-551; Santerre et al. Proc. Natl. Acad. Sci. U.S.A 78: 4339-4343,1981. 41 HLDNA86 107 Activation of Kinase assay. Kinase assays, A highlypreferred Adipocyte ERK for example an Elk-1 kinase embodiment of theinvention Signaling Pathway assay, for ERK signal includes a method fortransduction that regulate cell stimulating adipocyte proliferation ordifferentiation proliferation. An alternative are well known in the artand highly preferred embodiment may be used or routinely of theinvention includes a modified to assess the ability method forinhibiting of polypeptides of the adipocyte proliferation. A invention(including antibodies highly preferred embodiment and agonists orantagonists of of the invention includes a the invention) to promote ormethod for stimulating inhibit cell proliferation, adipocytedifferentiation. An activation, and differentiation. alternative highlypreferred Exemplary assays for ERK embodiment of the invention kinaseactivity that may be includes a method for used or routinely modified toinhibiting adipocyte test ERK kinase-induced differentiation. A highlyactivity of polypeptides of the preferred embodiment of the invention(including antibodies invention includes a method and agonists orantagonists of for stimulating (e.g., the invention) include theincreasing) adipocyte assays disclosed in Forrer et al., activation. Analternative Biol Chem 379 (8-9): 1101-1110 highly preferred embodiment(1998); Le Marchand- of the invention includes a Brustel Y, Exp Clinmethod for inhibiting the Endocrinol Diabetes activation of (e.g.,decreasing) 107 (2): 126-132 (1999); and/or inactivating adipocytes.Kyriakis JM, Biochem Soc Highly preferred indications Symp 64: 29-48(1999); Chang include endocrine disorders and Karin, Nature (e.g., asdescribed below under 410 (6824): 37-40 (2001); and ““EndocrineDisorders””). Cobb MH, Prog Biophys Mol Highly preferred indicationsBiol 71 (3-4): 479-500 (1999); also include neoplastic the contents ofeach of which diseases (e.g., lipomas, are herein incorporated byliposarcomas, and/or as reference in its entirety. described below underMouse adipocyte cells that “Hyperproliferative may be used according tothese Disorders”). Preferred assays are publicly available indicationsinclude blood (e.g., through the ATCC). disorders (e.g., hypertension,Exemplary mouse adipocyte congestive heart failure, blood cells that maybe used vessel blockage, heart disease, according to these assaysstroke, impotence and/or as include 3T3-L1 cells. 3T3-L1 described belowunder is an adherent mouse “Immune Activity”, preadipocyte cell linethat is a “Cardiovascular Disorders”, continuous substrain of 3T3 and/or“Blood-Related fibroblast cells developed Disorders”), immune disordersthrough clonal isolation and (e.g., as described below under undergo apre-adipocyte to ““Immune Activity””), neural adipose-like conversionunder disorders (e.g., as described appropriate differentiation belowunder ““Neural Activity conditions known in the art. and NeurologicalDiseases””), and infection (e.g., as described below under “InfectiousDisease”). A highly preferred indication is diabetes mellitus. Anadditional highly preferred indication is a complication associated withdiabetes (e.g., diabetic retinopathy, diabetic nephropathy, kidneydisease (e.g., renal failure, nephropathy and/or other diseases anddisorders as described in the ““Renal Disorders”” section below),diabetic neuropathy, nerve disease and nerve damage (e.g., due todiabetic neuropathy), blood vessel blockage, heart disease, stroke,impotence (e.g., due to diabetic neuropathy or blood vessel blockage),seizures, mental confusion, drowsiness, nonketotic hyperglycemic-hyperosmolar coma, cardiovascular disease (e.g., heart disease,atherosclerosis, microvascular disease, hypertension, stroke, and otherdiseases and disorders as described in the ““Cardiovascular Disorders””section below), dyslipidemia, endocrine disorders (as described in the““Endocrine Disorders”” section below), neuropathy, vision impairment(e.g., diabetic retinopathy and blindness), ulcers and impaired woundhealing, infection (e.g., infectious diseases and disorders as describedin the ““Infectious Diseases”” section below (particularly of theurinary tract and skin). An additional highly preferred indication isobesity and/or complications associated with obesity. Additional highlypreferred indications include weight loss or alternatively, weight gain.Additional highly preferred indications are complications associatedwith insulin resistance. Additional highly preferred indications aredisorders of the musculoskeletal systems including myopathies, musculardystrophy, and/or as described herein. Additional highly preferredindications include, hypertension, coronary artery disease,dyslipidemia, gallstones, osteoarthritis, degenerative arthritis, eatingdisorders, fibrosis, cachexia, and kidney diseases or disorders.Preferred indications include neoplasms and cancer, such as, lymphoma,leukemia and breast, colon, and kidney cancer. Additional preferredindications include melanoma, prostate, lung, pancreatic, esophageal,stomach, brain, liver, and urinary cancer. Highly preferred indicationsinclude lipomas and liposarcomas. Other preferred indications includebenign dysproliferative disorders and pre-neoplastic conditions, suchas, for example, hyperplasia, metaplasia, and/or dysplasia. 51 HFOXA73117 Production of IL-10 Assays for production of IL-10 Highly preferredindications and activation of T- and activation of T-cells are includeallergy and asthma. cells. well known in the art and may Additionalhighly preferred be used or routinely modified indications includeimmune to assess the ability of and hematopoietic disorders polypeptidesof the invention (e.g., as described below under (including antibodiesand “Immune Activity”, and agonists or antagonists of the “Blood-RelatedDisorders”), invention) to stimulate or autoimmune diseases (e.g.,inhibit production of IL-10 rheumatoid arthritis, systemic and/oractivation of T-cells. lupus erythematosis, Crohn's Exemplary assaysthat may be disease, multiple sclerosis used or routinely modified toand/or as described below), assess the ability of immunodeficiencies(e.g., as polypeptides and antibodies of described below), boosting a Tthe invention (including cell-mediated immune agonists or antagonists ofthe response, and suppressing a T invention) to modulate IL-10cell-mediated immune production and/or T-cell response. proliferationinclude, for example, assays such as disclosed and/or cited in:Robinson, DS, et al., “Th-2 cytokines in allergic disease” Br Med Bull;56 (4): 956-968 (2000), and Cohn, et al., “T- helper type 2cell-directed therapy for asthma” Phramacology & Therapeutics; 88:187-196 (2000); the contents of each of which are herein incorporated byreference in their entirety. Exemplary cells that may be used accordingto these assays include Th2 cells. IL10 secreted from Th2 cells may bemeasured as a marker of Th2 cell activation. Th2 cells are a class of Tcells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that inducedifferentiation and activation of Th2 cells play a major role in theinitiation and pathogenesis of allergy and asthma. Primary T helper 2cells are generated in in vitro culture under Th2 polarizing conditionsusing peripheral blood lymphocytes isolated from cord blood.

TABLE 2 SEQ Score/ cDNA Contig ID Analysis PFam/NR Accession PercentClone ID ID: NO: X Method PFam/NR Description Number Identity NT From NTTo HSIDU19 520372 11 blastx.2 NA/PO4 sp|Q9Y2C5|Q9Y2C5 100%  9 461COTRANSPORTER HOMOLOG. HPRSB76 526310 12 blastx.2 POTENTIALsp|O54827|AT5A_MOUSE 83% 112 570 PHOSPHOLIPID- 40% 2 364 TRANSPORTINGATPASE VA (EC 1 HTEIL66 520125 13 WUblastx.64 (AK015053) putativedbj|BAB29693.1| 51% 36 509 [Mus musculus] HSNAY92 526730 14 blastx.2ubiquitous TPR motif pir|T02214|T02214 100%  18 347 isoform Y - humanHSABG21 526366 15 WUblastx.64 (AK021539) unnamed dbj|BAB13840.1| 97% 413556 protein product [Homo 99% 48 416 sapiens] HSAXB32 520470 16 blastx.2CDNA FLJ20378 FIS, sp|Q9NX85|Q9NX85 72% 1029 751 CLONE KAIA0536. 52% 754692 HSAXB81 520471 19 blastx.2 CDNA FLJ20489 FIS, sp|Q9NX17|Q9NX17 54%772 458 CLONE KAT08285. HSLCU73 520237 21 blastx.2 PRO1722.sp|Q9P195|Q9P195 62% 767 600 56% 587 468 50% 622 491 76% 478 416 51% 414322 HYBAY77 520394 24 blastx.2 SIMILAR TO POGO sp|Q13537|Q13537 47% 4471 ELEMENT. HSAVP17 519846 26 blastx.2 CDNA: FLJ21463 fis,sp|BAB15071|BAB15071 69% 691 509 clone COL04765. 73% 828 694 HSIEA14520387 27 blastx.2 UNNAMED PORTEIN sp|Q9N083|Q9N083 66% 667 479 PRODUCT.58% 779 672 HSNAQ47 847453 28 WUblastx.64 predicted usingemb|CAB04264.1| 35% 95 784 Genefinder˜cDNA EST yk38g9.3 comes from this11111111 this gene [Caenorhabditis elegans] HSNAQ47 526779 64 blastx.2hypothetical protein pir|T21713|T21713 35% 99 788 F33H2.2 -Caenorhabditis elegans HODDN65 520348 29 blastx.2 UNNAMED PORTEINsp|Q9N083|Q9N083 67% 660 493 PRODUCT. 74% 743 663 HPEAD79 520202 30blastx.2 PRO2550. sp|AAG35515|AAG35515 49% 808 497 HTODK73 526021 33blastx.2 CDNA FLJ13348 fis, sp|BAB14566|BAB14566 80% 21 401 cloneOVARC1002127, 100%  567 707 weakly similar to 1 43% 4 189 61% 418 51993% 404 448 71% 433 474 HSAWA27 520302 37 blastx.2 PRO2550.sp|AAG35515|AAG35515 78% 531 418 55% 649 521 60% 422 336 66% 396 352HSFAH43 520399 38 blastx.2 HYPOTHETICAL sp|Q9Y5K0|Q9Y5K0 81% 9 239 149.0KDA PROTEIN. 89% 241 351 HFAEF57 534142 40 blastx.2 Hypothetical 15.8kDa sp|AAG17270|AAG17270 47% 601 425 protein. HEGAH43 532596 41 blastx.2(AL360078) bA530N10.1 emb|CAC17684.1| 100%  29 361 (novel protein) [Homosapiens] HAGDG59 534165 42 HMMER PFAM: short chain PF00106 182.2 232 7952.1.1 dehydrogenase HAGDG59 534165 42 WUblastx.64 (AF126780) retinalshort- gb|AAF06939.1|AF126780_1 96% 124 1023 chaindehydrogenase/reductase retSDR2 [Homo sapiens] HCUIN80 534128 45blastx.2 CDNA: FLJ21463 fis, sp|BAB15071|BAB15071 78% 825 562 cloneCOL04765. HAPPS89 532135 47 WUblastx.64 (AF010144) neuronalgb|AAC08737.1| 64% 819 658 thread protein AD7c-NTP 64% 809 657 [Homosapiens] 55% 600 523 45% 667 602 68% 668 603 70% 810 541 HFIUR10 53206050 blastx.2 CDNA: FLJ21463 fis, sp|BAB15071|BAB15071 54% 497 339 cloneCOL04765. 90% 523 494 HLDNA86 535730 51 blastx.2 (BC000828) Unknowngb|AAH00828.1|AAH00828 81% 45 218 (protein for IMAGE: 3455200) [Homosapiens] HLTEF12 532354 54 blastx.2 isocitrate dehydrogenasepir|S57499|S57499 98% 469 1188 (NADP+) (EC 1.1.1.42) 98% 206 475precursor, mitochondrial - human HCFBJ91 531960 55 blastx.2 CDNA:FLJ21394 fis, sp|BAB15056|BAB15056 68% 764 615 clone COL03536. 63% 877758 40% 654 520 47% 761 705 HHFHP90 534615 56 blastx.2 PRO2550.sp|AAG35515|AAG35515 74% 982 800 79% 1101 970 HLYCQ48 527757 57 blastx.2CDNA FLJ20489 FIS, sp|Q9NX17|Q9NX17 62% 755 471 CLONE KAT08285. HBJEL68531924 60 blastx.2 PRO0117 PROTEIN. sp|Q9UI85|Q9UI85 59% 774 634 72% 643611 HFPDE86 527728 63 blastx.2 PRO2822. sp|Q9P147|Q9P147 72% 737 516

TABLE 3 SEQ cDNA ID Contig EST Disclaimer Clone ID NO: X ID: Range of aRange of b Accession #’s HSIDU19 11 520372 1-666 15-680 AI056789,AK024903, AB020527, AL391194, AL391194, and AL391194. HPRSB76 12 5263101-727 15-741 AA447438, T09355, AB011138, and Y09954. HTEIL66 13 5201251-605 15-619 AC067727. HSNAY92 14 526730 1-597 15-611 R64076, AA730371,R27397, AU139244, H71069, BF089099, AC010877, AC005820, AF000994,AF000992, AF000993, AJ002730, AF132076, AC002538, AL031427, AL031294,AL031686, AC006038, and AL009177. HSABG21 15 526366 1-571 15-585AA421255, AU145857, AA374532, AK021539, AK023067, and AK021849. HSAXB3216 520470 1-1026 15-1040 AA635135, BF803143, BE273825, AW162314,AW410844, AW265468, AW020150, AL036896, AW275432, BG180320, AI732690,AL121039, AI702049, AA526542, AV732057, AW504667, AA601376, AI065031,AW162332, AU151751, AI251024, AI926102, AW975150, AV760560, AW963489,AW974923, N72509, BE677164, AI521525, AW021674, AA640305, AA595661,AL042667, AL042670, AA525753, AI590442, AI890297, AI924950, AA218684,AI754926, AA171400, AV758849, BE244308, AI078409, AW338376, AA577706,AI744963, AV683406, AW277000, AA425283, AW277093, AA557945, AW157424,AA533066, AA533660, AI254267, AW813775, BF880881, AW970588, AW975239,AA084320, AU147226, M77888, BE138520, BG059139, AV730440, AA324108,AL044701, AA493546, AW169183, AI732327, AW151247, AA179364, BE328286,AI174703, AW020094, AI962030, BE967607, BE140949, AI064968, BG029528,AV761486, AW272389, AV712092, AV760508, AA313025, BF589864, BE676765,BF575954, AW265006, AI801563, AI049999, AI547110, AW855527, AW963552,AA935827, AI791659, AI872229, AI653776, AV755245, AI567676, BE154381,AI242236, AI955447, AI031759, AV717475, AW518140, AV710695, BG152746,AA601674, AW082104, BG236484, AV729908, AW020612, BE049409, BF530611,AA847341, AI538404, AI745666, AA573067, AI613459, BG059314, BE139230,AW328331, N99245, AW327852, AW192930, AI090377, AA610381, AI434103,BE139673, BG223498, AV743869, AW576299, BF846619, AI281622, AA112864,AW272815, AI521019, AL138262, AW177869, AI473671, AV758870, AV757180,AI445699, BF725844, AI500563, BF834298, AU155104, BF668217, AW675677,AW409626, AL041375, BF243651, AI884404, BG250794, AI537368, AC016655,AC010419, AP000694, AP001725, AL109921, AP000359, AL031311, AL021707,L35532, AL136139, AL117381, AC002115, AC002369, AC010422, AC002470,AL034420, AC022432, AL109798, AC006483, AL136131, AL034422, AP000553,AL133215, AL353748, AC008892, AL132765, AL157817, Z93244, AC006994,AL031658, AC004242, AL365444, AP000239, Z86090, AL136304, AL022316,AC005540, AC004771, AP000095, AJ400877, AP001670, AP001705, AC004816,AL049776, AC005808, AL117348, AL157768, AL008630, AC003665, AL353680,Z98941, AL121914, U52111, AL121983, AL159970, AC008747, AC005091,AC005049, AC002553, AC004206, AC005940, AC003007, AB014080, AC008969,AL157877, AC008083, AL360227, AC007324, AC006511, AL353804, AB041992,AL009181, AC025593, AC007845, AC004884, AC005098, AC005179, AB023048,AL356801, AL050318, Z97055, AP001630, AF030876, AL049694, AC003969,AC008897, Z97056, AL158040, AC009086, AL050335, AL356215, AL109984,AP000514, AL135927, AC007227, AL117329, AL117258, AF168787, AC016026,AL133445, Z99716, Z93017, AL356379, AC003037, AC022402, AP002529,U47924, AC006974, AC008569, AC009244, AC005304, AC010789, AL136418,AC004890, AC002565, AP001717, AC008687, AC007688, AJ277557, AC005180,Z83844, AC006071, AL137818, AL035693, AC005726, AC006162, AP001748,AC004913, AL035072, AL132795, AP000501, AL132653, AC005841, AC005519,Z70289, AC011480, AL359457, AC006536, AC005838, AL109806, AL137783,AP000555, AC005274, AC005776, AL008582, AL353807, AP000511, AL031427,AC005736, AC008543, AL049712, AC002351, AC000115, AL133404, AL357374,AC005529, AC026776, AP000031, AL080243, AP000030, AC005937, AP001711,Z97632, AC005261, AP000252, AF205588, AL139824, AL122020, AC007055,AC008267, AP000211, AP000133, Z83840, AC006079, AL031846, AP000113,AC002073, AC007686, M89651, AL354720, AC010526, AC009134, AC011479,AC004826, AC011490, AF196972, AL136969, AC008068, AL022721, AL050349,AL096677, AC005527, AC008745, U91325, AC020916, AC007934, AC005484,AL121712, AC067969, AC024561, AC011495, AP000552, AC008784, AC005793,AC010326, AC011444, AL008732, AC004150, AF111168, AL138713, AF031075,AL035659, AC009314, AC010553, AL031347, AC005324, AC008536, AC000025,AL109797, AF141309, AC011442, AL358777, AC007225, AC008403, AC007272,AC007386, AC006060, AL157372, AL138759, AC004997, AC010271, AL135905,AC008753, AF015720, AP000503, AF134726, AC005829, AL031729, AC005913,Z95116, AL138807, AC002991, AF217403, AF196779, AC005531, AL137139,AC005632, AC002395, AL022336, AP001973, AC027536, AC011957, AC010419,AC010419, AC010419, AC016655, AC016655, AC016655, AC002369, AC002369,AC018548, AC018548, and AC018548. HPEAD48 17 520367 1-611 15-625AW964468, D80045, AW966389, AW949645, AW966330, AW949642, AW964532,AW965158, AW949643, AV738340, AV744012, AW975618, AV744690, AW366296,AV702035, AV723097, C14389, AV724520, AV742048, AW962395, D51799,AW964488, AV719468, D59502, AW375405, D80195, AV720791, AA305578,AW966050, AW965185, AW965197, AW959597, AV742732, AV718800, D80164,AV742050, AW966053, AW966013, AW949658, AW975621, AW966054, AV719783,AW966534, AV720464, AI905856, AW960465, AW949654, AV705134, AW966022,AW966075, AW966041, C15076, AW966065, AW973541, D80038, AW978648,AW959062, AV719324, AV718440, AV720028, AW975613, D59467, AW965196,AW965184, AW965175, D59275, AV718770, AW966030, AV719188, D80227,AW964477, AW966062, AW949641, AW949646, AW959570, AW966029, AV718707,AV705869, AV699927, AW973334, AW966531, AW978634, AW956434, D58283,D80022, AW965163, D80166, AW959799, AW966059, D80193, AW960473, D59859,D59619, D80210, D80391, AV704180, AW960553, D80240, C14331, AW973474,AV719822, AV718692, D59787, D51423, AW973488, AW978661, AV720211,AV718844, D81030, D80253, AV718489, AV720203, AW964756, AW973307,D80043, AV723927, AW177440, AV718938, AW949656, AV718633, AW959628,AW965177, AW975605, AW973485, AV718931, AV720878, AV719557, AV720731,AW973482, AV699447, AW958992, AW958993, AV722801, AW959136, AV699550,AW962082, D80269, AW959202, AV720812, D80212, D80196, AW949657, D80188,D80024, D50979, D80219, AV706147, AW753067, D80268, AW966043, AW949655,AW959582, D59927, D59610, AV720533, D57483, AA305409, AV719628, D80378,D80366, AV741220, D51022, AV706229, AW973330, AW962245, AV721386,AW973447, D59889, D50995, AA514188, D81026, AV727978, AW960454,AW949653, AW949631, AW949618, AW959469, AW964737, AW966032, AV720150,AW960532, AW956397, AW949629, D51060, AW949633, AW949632, AV700889,AV699866, AV701123, C14429, AW973490, AW752082, AV726812, AW753053,AW965176, AW966023, D80241, AW177501, AW960564, AW177511, AA514186,AW178893, AW949630, AW960504, AV699746, AW375406, AV700229, AV699682,AW973465, AW960474, D80248, AV718530, AV703542, AV701004, D80522,AW975623, T03269, AV720654, AV699669, AW960570, AW360811, AW179328,AW964541, AW973473, AW973445, AV720220, C75259, AV720616, AW966332,D80251, AV719000, C14014, AW352117, D80133, AW960514, AW966399,AW966333, AW966331, AW753041, AV718908, D58253, AV742001, AV742667,AV742022, AL136136, A62298, A62300, AX047063, AX047064, AR070327,A82595, AX047062, A84916, AX020191, AX035434, AR018138, AX020190,Y17188, AR016808, AR074545, AX027925, AJ132110, AX033851, AR092424,A94995, AF058696, AB028859, AR008278, AX021518, AR087649, Y17187,X67155, D26022, A25909, AX028130, A67220, D89785, A78862, D34614,AJ302649, Y12724, AR016514, D88547, AR077702, A43190, AR060385, X82626,AB002449, AR025207, AR008443, AR038669, I50126, I50132, I50128,AR091537, I50133, A30438, AJ294956, AR066488, AF260572, AR060138,A45456, A44171, A26615, AR052274, AX015396, AB012117, I19525, AR074139,Y09669, A43192, AR066490, I14842, AR066487, AR074136, I18367, X68127,AR054175, AX014811, AJ287395, A85396, AR074141, AR066482, D50010,AR088705, AX042372, A85477, D88507, A86792, A63261, X93549, AR008277,AR008281, A70867, AR008408, AR062872, AR093385, I79511, AR016691,AR016690, U46128, A64136, A68321, D13509, AR060133, AR050070, AF135125,AJ000347, I32384, U79457, AF123263, AR032065, and AR008382. HPVAB94 18526749 1-805 15-819 BF370024, AW449056, AF131217, AL163247, AF165147,AF131217, and AF165147. HSAXB81 19 520471 1-768 15-782 BF367762,AA904211, D29019, AA469451, BF804385, AL079734, AL353771, AL133245,AL031659, AC005520, AL121653, Z49236, AF196779, AL031681, AJ295844,AC008379, AP000513, AL135744, AP001726, AC015651, AL079342, AC004019,AP000694, AL034372, AC010553, AL109804, AP000359, AC007688, AL021407,AC004824, AF254983, Z84469, AC004983, AC002418, AL109984, AC011469,AC011742, AC004386, AC008753, AC005089, AC007773, AF168787, Z85987,AL132639, AC006483, AL353596, AL049829, AF196969, AC004898, AC027319,AL163201, AL136170, AC007845, AL035458, AL138717, AP001748, AC004491,AL033529, AC007404, AC009958, AL445472, AC021016, AC007731, AL031282,L48038, AC006480, AC005261, Z82202, AC009194, AL021391, AC011464,AC005500, AL049776, AC004106, Z82190, AC008649, AL109936, AL022476,AC005954, AC011489, AC007383, AL023583, AL109743, AC004975, AC009086,AP001760, AC009408, Z82214, L78833, AC011248, AC002115, AC000052,AL035681, AL109797, Z84466, AC008745, AC008760, U82671, AC007298,AL139343, AX039602, AP000095, AL022163, AC004882, AC009298, AC005512,AC019213, AC019213, AC019213, AL353771, AL353771, AL353771, AL354721,and AL354721. HSAYC21 20 526188 1-641 15-655 BE856451, AA653742,AC008372, AC000353, AC011097, AC011097, AC011097, and AC019279. HSLCU7321 520237 1-784 15-798 AI866487, AV759172, AW965008, AA704393, AW440545,AL119691, AI963720, AL135377, AI282479, AA587604, BG109996, AI745151,AV762957, AI732151, AW103758, AW088846, AV710066, AV735495, AW028429,AA167055, BF592311, AV742057, BF826830, AW963463, AV728425, AI270117,AW504104, AV734149, X87344, AC006512, AC022392, AL445490, AL159168,AL355094, AC002553, AC006328, AC019187, AC012064, AL353706, AC006011,AC015651, AC007690, U73634, AC005630, AC010724, AC005899, AC010725,AP001714, U50871, AC004815, AL109921, AL445217, AC009399, AL133453,AL162390, AP000555, AP001717, AC006482, AP000048, AF165926, U85195,AL049776, AC009403, AC005098, Z81369, AP000553, AE000658, U95742,AL079295, AL133353, AC004166, U96629, AP001711, AC005512, AC003983,AL109798, AC006449, AL135927, AC007227, AC004996, AC003029, AF129756,AC004824, AC005844, AC020629, AL136526, AC022078, AL136418, AC007386,AF217403, AL109758, Z84469, AC011742, AC005971, AC011475, AL050308,AC005907, AC004858, AP000037, AP000105, AC004890, AC011497, AL136979,AP000557, AC005089, AC009307, AC007216, AC008379, AC005257, AL109935,AL022322, AC008115, AL033529, Z84488, AC000025, AC005784, AL079335,AC011484, AC007842, AC004019, AC023511, AL022328, AC009086, AF219991,AF196779, AC009228, AC005104, AL022320, AP000114, AP000046, AL009183,AC006285, AP001710, Z94044, AC002996, AC005231, AL031588, AC022311,AL132639, AC006013, AL035587, AC025593, AC008569, AF053356, AC011811,AC009244, AC005020, AX039602, AC006515, AC011445, AC000026, Z93015,AC010267, U91321, AL136365, Z86090, AB017654, AC007546, AC006211,AC006001, AC027319, AL022327, AC007536, AC011464, AC004971, AL133232,AC018639, Z93930, AC004526, AF131216, AL138759, AL031733, AC004644,Z82208, AL133174, AC009530, AC005280, AL137818, AC004686, AP001053,AP001670, AC007242, AL160175, AL021391, AL135749, AC005722, AL035249,U47924, AC007055, AL118520, AL161670, AF207550, AL137119, AC020906,AC005696, AC006950, U91323, AL096701, U95739, AC006275, AC005924,AC004257, AL121655, AL049795, AL035684, AC008745, AC011495, AL109804,AC004967, AP000509, AF064861, AF109907, AC006115, AL080243, AC000052,AP001695, AC005632, AC006511, AF134726, AC004802, AF196972, AL049759,AL158830, AF196969, AC016027, AC011527, AC006121, AL163279, AL031681,AL121893, U80017, U95090, AL022343, AL109914, U62293, AC016830,AC002470, AC005081, AL022316, AC002544, AC005261, AC011470, AL021393,AL359238, AC005988, AC006538, AC009475, AC002059, AL109743, AL034380,AC004253, Z93017, AP000961, AC010422, AL049589, AL031281, AC008764,AP001688, Z93020, AL135839, AC009311, AC006077, U07561, AC005056,AL136137, AC010311, AL138784, and AL138784. HSSFZ70 22 526499 1-63215-646 AI207437, BE877529, AI567204, AI478230, Z98486, BE908499,AW827226, AV705400, BE880328, BE873944, AI005023, AI953563, BF724930,BF032694, AA654003, BE875705, AA292238, AV702832, AV704592, AV702861,BE873725, AW410260, AW410829, AI560202, BE907715, BE881031, BG176797,AA806061, AA166890, AI564171, AL037085, BG177029, AW088118, AW411537,AI625499, BE878269, AI919534, AL037693, AA088891, BE877210, AA225922,BE874251, AA575974, AI963858, AI609179, AI634594, AA180745, AW601571,BE880300, AI699885, AA766508, BG165872, AW103371, AV722918, H29578,AI289160, AA018554, AI888869, AI579931, BE257077, AI799374, AW162189,AI625489, AW409568, AA088755, AA565315, AV710589, BE875112, BF672427,AI743700, AI699797, BF983585, AA283870, AI650301, AA019432, AW411050,BE908424, BG179111, AI913315, AA844225, AI654329, AA575854, AI568346,AI926112, AI565406, AA193247, AI689674, BG253912, AI619781, AA931604,AI630615, AI769728, AB048935, S77771, Y14485, AF114170, AF130470,AL049382, AF081366, S69385, AF093542, AF247674, AK026784, L00972,AL096751, AC002467, AC002401, AF110520, AP000839, AP000839, AP002833,and AP002833. HTEIP36 23 520468 1-738 15-752 AI695417, and AC037454.HYBAY77 24 520394 1-801 15-815 U49973, AC006080, AL353802, AF031076,AF031078, AF030876, AL022318, AF178030, AC006398, AL034410, AL049176,AC007380, AC010967, and AL031848. HROAE78 25 520365 1-864 15-878AC022509. HSAVP17 26 519846 1-836 15-850 AV758870, AI282479, AW855643,AW275432, BE148969, BF946053, BE138594, AW974932, AA704393, AW021917,AA630854, BE062478, AI361090, AV760915, AA456924, AV760508, AA536040,BE148935, BF675251, AV761107, AA829036, AI609972, AV760014, BE062476,AW806990, AI278972, AW271904, AV695478, AW502237, AW970958, BF681619,AA598927, AA757661, AI888468, AI978782, AW969941, AW851405, AI362442,BE138484, AL119247, BF991208, AV756491, AA524616, AW026305, AI421950,BF772474, AI419337, BF811714, BE062159, T74524, AW327624, BE062484,AW383952, BF589824, AW958962, AL037714, AW272294, AA468131, AI635028,BF804359, AI358712, BE178231, AV754716, AA558404, AI380617, AI912401,AC005086, AC005527, AF047825, AC004964, AC000025, AC005103, AC005529,AC007383, AC008747, AL137818, AP002028, AC002429, AL159168, AC005082,AL139095, AC011452, AL117381, AC002115, AC008812, AP002085, AC000035,AL137800, AL135927, AC007227, AL121653, AC001231, AC004876, AC005995,AC002418, AC004778, AL031589, AL139100, AC007193, AC006967, AL021918,AL136981, AJ400877, AC007263, AL121895, AC007395, AL035462, AC016526,AP000193, AL355392, AC005080, AC003029, AC002119, AL117356, AL161445,AL031311, AL121897, AL135744, AP000117, AL121753, AC011465, AC008102,AC007707, AC004522, AC023510, AC010412, AC009004, AC006241, AC018712,AL049766, AC003043, AC005755, AC007912, AP001718, AP000313, AP001748,AC005257, AC002996, AC020898, AL009179, AL133174, AL133289, AC021016,AC025435, AC010627, AP000050, AC002369, AC004386, AC004966, AC005933,AF109907, AC010422, AL160191, AL133545, AC004865, AL121890, AC024078,Z82172, AC005545, AL158040, AC004253, AC005924, AB045146, AC008745,AC004847, AL049699, AC021999, Z95331, AL022316, AC005088, AL049569,AC020754, AP000501, AC008379, AL121920, AC007055, AL034549, AC020893,AL133245, AC006077, AC006071, AC018639, AC007386, AL096701, AL138756,AC009470, AP001631, AC004148, AC005400, AC002364, AC002425, AL022326,AC005236, AC002553, AC005620, AL035086, AC008651, AL133332, AC009086,Z83844, AL022323, AC003006, AL135902, AC003101, AC008641, AL034429,Z85986, AC025588, AC007283, AC005057, AC020917, AC005519, AC074338,AC021036, AC027319, AC005480, AL136137, AL138878, AF207550, AC010458,AC002314, AC015853, AC004089, AC008403, AC006277, AC016995, AL159158,AC000069, AF217403, AC005696, AC006117, AL009181, AL354864, AC004989,AL021397, AF307337, AC008753, AL008718, AL022163, AL109976, AC004560,AL121601, AC011508, AC005004, AL357153, AC009244, AC004686, AC007956,AC005971, AC083863, AC002350, AC004884, AC002404, AC002549, AL136972,AC005058, AL035423, AC005594, AL049653, AC011464, Z93015, AC005701,AC005081, AF001552, AC008521, AL024498, AC024084, AL078645, AL136365,AL133294, AC004659, L31848, AL022320, AJ133269, AC004662, AC005666,AP001728, AC007450, AC003950, Z75887, AC002504, AL031588, AL136418,AP000350, AP000547, AL138735, AC008616, AL122001, AC022516, AF124730,AF001549, AL049757, AC008738, AC007066, AC008736, AP000557, AL035454,AC007326, AC004819, AL049748, AL008582, AC004099, AL033527, AL031282,AC005940, AL138976, AC011742, AL008721, AC010633, AC010633, andAC010633. HSIEA14 27 520387 1-774 15-788 AW301736, AL042667, AL042670,AV695478, AW328202, AW238341, AI538491, AA569494, AA572983, W42588,AI278972, AL120141, AI821382, BE042006, AI307201, AA501461, AA837597,AA514450, AV761433, AI753969, AA613761, H68343, BE139139, AI250552,AV648653, AI284543, AW020612, AI689198, AV656063, AW613448, AI005613,BF917473, AI254770, AI744830, AI820534, BG030789, AI251284, AI251034,AI251203, AI223626, AI249853, AA526099, AW023515, BE138387, AA602105,AI798407, AI439393, AI690497, BE169870, AI692245, AL133519, Z83819,AC002350, AC021036, AL031733, AP001706, AC005844, AF017104, AL157372,AL117341, AP000073, Z84487, AC007282, AC011742, AL022336, AC019106,AC008011, AF049895, AL109797, AP001710, AL160231, AP001759, AL137073,AL391114, AL049713, AL133454, AP001683, AC058791, AP000104, AC007880,Z93341, AC011904, AP001714, AC008738, AC016689, AC008543, AC009225,AL024507, AB000882, AC006449, U96629, AP000344, AC007225, Z98946,AL050333, AL049838, AL121782, AC004491, AL009181, AL035249, AC005064,AL121825, AF205588, AC024082, AC009060, AC005663, AL121949, AC007546,AF107885, AP002530, AC011500, AL137060, AC005969, AC005216, AL031848,AC005291, AL133453, AL353802, AL078591, Z98742, AC002418, AC004386,AP001728, AC013429, AC018513, Z83843, AC002303, AL139826, AC005703,AC008008, AC003042, AL033381, AC005332, AC007938, AL121872, AC006121,AL138755, AC073574, AC011311, AL139109, AE000658, AP001725, AL138828,AC009236, AF196971, AC002429, AC009016, AC012156, AL157791, AC004847,AL109743, AC006160, AP001712, AC007486, AL157413, AC005352, AL109923,AP000692, AC005386, AC004977, AL034550, Z96050, AL360227, AC000004,AJ229041, AL121878, AC008267, AC025442, AL117333, AL136979, AL050317,AC008750, AL359704, AL031681, AF027390, AC004743, AL008732, AC009501,AC007690, AC005562, AC078889, AL355886, AC009238, AC005214, AF280107,AL121852, AC009470, AC007773, AP000352, AL031685, AL136365, AC005821,AP001720, AC020898, AL356379, AL049760, AL049776, AL441887, AC010219,AC003992, AL138752, AC012351, AC012309, AL080317, AC002381, AL035458,AL031289, AC003662, AC036103, AC007312, AL445687, AC006120, AC024600,AF241726, AC005358, U80017, AC004859, AL133153, AC027689, AC005069,AC007376, AC009953, AC018684, AC010363, AC007868, AC004833, AC004000,AL031427, AL022318, AC007240, AC003108, AC010328, AL132777, AC007542,AC006455, AL157827, AC006001, AC007637, AC007262, AF015720, AC006996,U91323, AL158167, AC004745, AC074331, AL121585, AC006372, AC009455,AF190464, AL035555, AC022493, AC003029, Z84474, AC016543, AC027644,AP001660, AC008518, AL391259, AL049834, AL121652, AJ239323, AL163853,AC002289, AC005488, AC004887, AC005690, AC007000, Z98950, AL109655,AL133519, and AL133519. HSNAQ47 28 847453 1-824 15-838 BG031135,BE882847, AA508130, BF897342, N84247, BF793144, H56425, BF735575,BF735202, BE734983, BF735512, BF735472, BF735739, BF735568, AI281168,BE543895, R86099, BF735124, H25599, BF991203, H44341, R70461, AI273738,BE830524, BF735219, BF734982, AI221561, AB007962, AC018593, AC018593,AC025992, AC025992, AL162612, AL162612, AC024468, and AC024468. HODDN6529 520348 1-741 15-755 BF526964, AV734149, AV760019, AI246796, BE063437,AL135377, AI061313, AA515048, AW274191, AI306232, AL046519, AI251576,AI248050, BF828714, AI311647, AW973992, BF826830, AI207465, AW505253,BF340002, AA303007, AW243793, AA704393, T05118, AI583466, AA504818,T74524, AW855643, AW468048, AA737309, AW732205, AI270177, BF821897,AV755654, AW504168, BG222813, AW965008, AI085242, AW516080, AW500684,AL079734, BE077105, AI380617, AI499954, BE062478, AW237905, AA806804,AA484201, BE148969, AV703187, AI612142, BF724699, AA513551, AA730305,AA515728, AW970940, AI491755, BE301584, AW963444, AI192440, AI053827,AV741663, AA524616, AA515723, AW975626, AA827383, AA678950, BF447461,AW963463, BE138594, AA484366, AI610941, AA829036, BF811714, AW407632,AA569089, AI583252, AA502532, AW502873, BF990660, AW969941, AL046471,BF821009, AC006111, AP000553, AC004644, AC005225, AC005484, AL354889,AC006483, AC005229, AL138759, AC008102, Z84468, AC010202, AL022311,L78810, AC004851, AL008725, AC015550, AF006501, AP000075, AL022165,AC010326, AC007151, AL031685, AC006974, AC011530, AC005821, AC011462,Z97876, AL049835, AL122001, AC004448, AC004686, AC009247, AC006388,AC009311, AC004821, Z99716, AC005480, Z86090, Z98044, AL133387,AL035462, AL049569, AC007249, AC010363, AC002400, AL109930, AL121675,AC008641, AL121845, U63630, AC002312, AJ003147, AC004234, AL035086,AC008072, AL031587, AC007620, AL109963, AL133286, AL049766, AC006077,AL022238, AL137139, AF107885, AP002085, AL033529, AF254822, AC005793,AC004000, AC006480, AL445248, AC005089, D89013, AL034369, AC005071,AC011442, AC005940, AC008736, AL353701, AC004382, Z93015, AP001330,AC006312, AL034420, AP001748, AC005015, AL034424, AC006512, AP001629,AF001552, U08988, AL031847, AL136300, AC005972, AC007739, AL023807,Z82215, AC011490, AL118501, AL022069, AP000065, AP001752, AC004971,AC006344, AC005098, AC000379, AC011811, AC004895, AL034380, AC004832,AC002352, AC008760, AC010150, AL034421, AC004876, AL109936, AC015853,AC012384, AP001759, AC010519, AL163208, AC005971, AL031282, AC009086,AC007276, AL137072, AC008066, AC004622, AL133480, L78833, AL020993,AC004542, AC004494, Z83838, AC006549, AL109976, AC006262, AL022336,AC004966, AL022322, AC006057, AC007384, AL161659, AP001631, AP000555,AC008745, AL031727, AC008115, Z93023, AL096701, AL080317, AC004477,AL122020, AC010524, AL121899, AJ009616, AL034562, AP001435, Z93241,AC005358, AC004867, Z82244, AC004797, AL121753, Z98949, AC005377,AC004143, AF190465, AC019155, AC016025, AL135901, AP001754, AP001728,AC005488, AC003963, AC005602, AL133553, AC006449, AL035252, AP001610,AP002535, Z93244, AC002563, AL133174, AL034417, AC011495, AC009516,AC007676, AC004084, AC008474, AL034548, AL031118, AL137162, AL049829,AL138878, AC015651, AC006441, AC005666, AL078474, AL035071, AF064861,AC008636, Z94056, AC006285, AF038458, AP000295, AL031255, AL133153,AC004929, AC004778, AC010618, AC022392, AC018751, AC010789, AC022436,AC005756, AL096773, AL049643, AL109804, Z97985, AC073539, AC008675,AC018757, AL158830, AP001715, AL133240, AC000097, AC025809, AC005747,AC008910, AL133545, AC11491, AL138741, AC006547, AL121893, AC004796,Z97056, AL139100, AC008403, AC022274, AL049712, AC004089, AL136162,AC073052, and AC073052. HPEAD79 30 520202 1-799 15-813 AC004590,AC069275, AL117382, AC002094, AC009955, AC055740, AC011470, AC004965,AF109907, AL109804, AC008745, AL121653, AC018832, AC018738, AC009502,AL136137, AC016543, AL096701, AC011449, AC006345, AC007637, AC003029,AL050341, AC008403, AL161670, AC008764, AC023472, AC006449, AC005632,AC005041, AJ011930, AL163300, AL034405, AL109865, AC004962, AL096814,AC007666, AF053356, AL109897, AL080314, AC013434, AC005971, AC009756,AC007722, AC008474, AP001627, AC005089, AC021999, Z83844, AC004659,U07562, AC007793, AL158846, AL158846, and AL158846. HRDED19 31 5260191-499 15-513 AI685811, AC040946, and AC021221. HSAYS89 32 526621 1-56215-576 AC011319, AC069204, and AC069204. HTODK73 33 526021 1-100515-1019 AI347130, AW027513, AW954660, AI660559, BF063427, BG230588,AI340321, AU155474, AI214222, AW006987, AI803717, AW301687, BE221184,AW016409, AA018238, AA019155, AI336534, AA015924, AI283525, AA921711,AA307210, AI090373, AA878131, AI269256, BF761125, AA017114, AI857978,BF229860, BG030487, AI368031, H84864, R8448, AW403382, AI367923,AW900223, AI284356, AI080385, AI350788, F29712, BE251142, AL536301,AA987803, BF761104, BF808782, AA017329, AA018635, BF593228, AA534529,BF851700, AL536302, W21988, BE910304, AA280994, AB031051, AL357033,AK000551, AF104334, and AK023410. HSVAM10 34 520328 1-419 15-433AI654853, AC021164, AC021164, and AC021164. HSNBN57 35 526721 1-62815-642 AA505101, AC007263, AL157736, AL357093, and AC007263. HSVBD22 36520557 1-653 15-667 AL138996, AL138996, and AL138996. HSAWA27 37 5203021-640 15-654 AW021917, AI753488, AI040051, AI499954, AA809546, AA584489,AW969743, AW516304, AW270385, AI679413, C06046, AA483606, AW338021,AW500250, AI753113, AA570740, AA315361, AW963444, AW243793, AA070899,AW105729, AW083934, AA613627, AV649853, W95976, AW341903, C75406,AW167154, BF764092, AA568204, C75574, AI598003, BF871805, BF673854,AI004591, AI002952, AA568314, AA610433, BF850062, AA491821, AA488746,H18120, AI702049, AW068316, BF030278, AI590499, AW957372, T50676,BE019467, AA878140, AI754955, AP001707, AC004707, AC005046, M37468,AJ277546, AC004846, AC008482, AL121656, AC000025, Z99716, AP001760,AP000042, AP000110, AP001716, AP000338, AC005202, AC005969, AL136124,AP000216, AL354864, AC005924, AC006329, AL031846, AC027319, AC005913,AP001714, X02571, M12901, AC004386, AC008556, AL022336, AC026776,AL008735, AC005291, M91159, AC005778, AC004236, AL157760, AP000104,AL135927, AC007227, AL118520, AC005840, AC005000, AC004000, AC011742,AC006511, AC004971, AC008403, Z83826, AC008848, AC010328, AL035420,AC007383, AC004686, AF069291, AL022237, AL132659, AC004887, AL031286,AJ009615, AC018644, AC022148, AL031680, AP000555, AL117381, AC011490,AC005881, AC004531, AC005755, AL078584, AP000556, Z99128, AP000557,AC004840, AC012309, AC008072, AC007537, AL365505, AC004859, AP000289,AC007919, AC020904, AL136418, AP002015, AC007488, AL049830, AL161665,AC023490, AL050341, AC005971, AC007051, L78833, AC002301, AL049872,AC005074, AC007597, AC002369, AC005071, AL353602, AC010789, AC015651,AC003029, AC004650, AC000003, Z81450, AC004929, AC005280, AL121655,AC006345, AL158830, AC004476, AL135783, Z98172, AL050335, AP000692,AP001666, AL121897, AP001712, AL353194, AC006126, AC004841, AC010422,AC008068, AL078590, AP001725, AC002418, AC006468, X74984, AC008747,AC008521, AL031255, L78810, AL133391, AC000159, AC007731, AL031284,Z98884, AC005500, AC018738, AC005839, AL021391, AC008784, AC009086,AC025588, AL353802, AC006459, AC016395, AC000379, AC004263, AL122001,AL033520, AC006130, AL133448, AC006441, AC006038, AC006484, AC004966,AF215937, AC005730, AC006057, AP000553, AC005180, AC008745, AL035071,AC002985, AC004975, AL109627, AC007957, AL050308, AL096861, AC004491,AL031587, AC005703, AC007421, AL022316, AC005598, AC011495, AC068658,AD000671, AP000512, AF023268, AC040171, AC010618, AC005484, AC005821,AL133245, U78027, U80017, AC007201, AP001710, AC005529, AC022243,AC022243, AL358353, AL358073, and AL358073. HSFAH43 38 520399 1-71715-731 AL038713, AI818151, AL133942, BE728951, AW157413, AA085707,AF074627, AU146974, AI925647, AL044349, AU145383, AI367384, AI887321,AF063514, BE549207, AA902828, AV719347, AI685116, BE646447, AU122382,AV726924, AL041411, BE409924, AU121228, AW847204, BF032731, BF681274,AU157470, BE385675, AI559442, AA864823, AF074693, AW852540, AW084901,AA767353, AW852539, AL133889, AI264673, AI858607, AL534431, AW139132,AA659014, AI610776, BE879973, BE165748, AI568919, BG059728, AU148220,AA745961, BF839073, BE893036, BE161913, AI924175, BE065468, AW177120,AW085676, AU143935, BF802808, AA581433, AI819528, AI250812, AI114529,BE065692, W58442, BF744176, BE745970, AI189033, BF953914, AI817158,AA932087, BE155605, BE065645, BE176566, BE065599, AA493735, BF760188,BF819519, BE179562, AI147839, AI749571, AI827133, AI675848, AA130536,AA055654, BE179906, AI132962, AW963409, BF760502, N24958, BE079382,AI376984, AL119355, BE267153, BG169215, AA493998, AI499286, AL523955,BG059067, AI733728, AA130476, AI453790, AA778304, BE079397, AW177237,AA174085, AA663566, BF963854, BF967102, AW074001, AW813858, AW795421,AI082077, BF869662, AW089655, AA846188, AA189081, AI133073, AW262471,BG010863, AW419031, AI862874, AA287329, W85828, BF806618, BE065644,AI973178, BE145271, AW177226, T16214, BE044603, BF854640, AA129986,AF063501, AI051341, AW995608, AW938300, R80440, BF576607, BF056069,BF915223, AW090210, N64574, AL521095, AI439860, BF878385, AV732745,BG235936, BE179619, AW177231, AW177317, W49501, AW102963, BE065597,BE154392, AW947245, BF935745, BE155635, AI610326, AI811854, AA807609,AA843874, T57073, AW571697, AW962610, AI984510, AA157033, AW440317,AI246569, AI027421, BF689260, AW813744, N26540, BG230953, AI921101,AW167452, AI983921, BF339110, AV764113, AI927861, AA121916, AA099788,AV661095, AW235478, AW847202, AW023397, H97952, AW847178, AA092309,AW151307, AU145663, AU118990, AA501873, AA984116, BF943200, AW945630,AA709024, AW889871, BF958183, AI557354, AU144339, BF751284, BF888049,AI832184, AW177266, AI952804, AI862212, BF843702, AI479035, N76274,AV729448, AW827404, AW517766, AA911409, AW272376, BF821660, AA811111,AI025602, AW833263, AA854527, BF946785, AV730063, H20876, BE154996,AA889273, BE538852, AI439415, AU143906, AI369914, AI561208, AW948678,AI538654, T62931, AI811494, AA581340, BF946782, AW193992, AA515372,N20521, BF760191, AW833302, AW948681, AA166854, AI346802, BF873305,N90055, AW168798, AW948679, AL139348, U73465, AC046130, AL021408,AL355305, AC068130, AC021070, AL157915, AL391071, Z82193, AL136080,AC008041, AL133370, AC009481, AL031114, AC005740, AL161743, AC021998,AC019100, AL121933, AL035453, AL078601, AL034408, AL078644, AB045365,AL121882, AL132826, AC026201, AL353745, Z93015, AL035689, AL133417,AF241733, AC000112, AC008929, AC002066, AF241726, AL356596, AL031684,AJ133269, AL157775, AC005341, AC005000, AC016607, AL033379, AC013458,AC018360, AC007514, AC009501, AL049734, AL050309, AC006600, AC003686,AL008627, AP002535, AL049778, AC007251, AC004385, AL354891, AL139093,AL135978, AC011478, AC007744, AL358214, AC005146, AP000173, AL163194,AC008572, AL121932, AC001608, AL157694, AL109750, AC008116, AL109759,AL138773, AC060232, AL139232, AL161730, AL138696, AX015914, Z92542,AL109800, AC002519, AL121879, AL079303, AL024507, AL390965, AC063956,AF130249, AL023806, AC002402, AC018468, AL158812, AC017067, AC018504,AC026184, AC008818, AC023118, AC068313, AC018492, AL109620, AL135940,AC061958, AC009241, AC008134, AL139009, AF241727, AL133381, AC067744,AL356750, AL133409, AL138810, AJ298105, AL033403, AC060834, AC023430,AF207550, AC008583, AP001721, AC004103, AJ277546, AC022274, AC003668,AC006226, AC007000, AL139350, AL133284, U69729, AL031655, AF307337,AL022577, AP000333, AL356982, AL034396, AL035464, AC009289, AL122016,AC007276, AL158141, AL110505, AC010738, AC005191, AC011230, AC083875,AL136970, AC005284, AC006516, AC068799, AL161788, AC006556, AC004384,AC004388, AC023478, AP002026, AC023483, AL163206, AL161916, AC008861,AL133462, AL009172, AL365214, AC006313, AC008598, AL360088, AC034195,AC007570, AC024947, Z95437, AP001342, AL391260, AC020717, AC006010,AL035700, AL139109, AF238375, AC005164, AL078615, AL022394, AP001821,AL157830, AC010276, AC011507, AL450448, AC007358, AC003051, AC024900,AL163152, AC024600, AC005213, AC006581, AC073102, AF027390, AP000068,AL157777, AL359703, AC004111, AL139014, AC002379, AL392106, AL359854,AC005214, AL356800, AF130343, AC007486, AL133514, AL450169, AC006352,AC022428, AC006143, AF238380, AP001677, AC007392, AP000112, Z95400,AL133411, AL034407, AL022171, AL133247, AC000120, AC009401, AL392108,AL158040, AL158015, Z82216, AC006984, AP001538, AL109804, AC008174,AC021863, AC009948, Z72001, AP001660, AL031653, AL109809, AL355540,AL031075, AC005019, AL354943, AC026197, AC005138, AL137012, AC022360,AL139813, AC012519, AC005358, AL034375, AP000340, AC011594, Z97987,AC004848, AL031726, AL031726, AC023412, AC058811, AC008041, AC008041,AC011739, AL356513, AL356513, AL109804, AC006226, AL079303, AC010192,AC010192, AC069443, AC069443, AC069443, AC006357, AC006357, AC026494,AC026494, AL121879, AC008134, AC008134, AL022165, AL022165, AL022165,AC021329, AC027116, AC027116, AL022395, AL022395, AL022395, AC005000,AP000445, AP000445, AP000445, AF207550, AF207550, AL138810, AL138810,AC007436, AF011889, AC005740, AC005740, AC004958, AC024947, AL031601,AL031601, AC007570, AC007570, AC013708, AC013708, AL157762, AL157762,AC007338, AC007338, AC018845, AC018845, AC068139, AL022394, AL022394,AL162390, AL162390, AL162390, AC011086, AC025802, AC025802, AC025802,AC069153, AC069153, AC007623, AC007623, AC016119, AC016119, AC018494,AC073487, AC073487, AL139109, AC025624, AC025624, AC025624, AL355075,AL355075, AC011435, AC011435, AC012321, AL355305, AL355305, AC009266,AC000120, AC000120, AC002060, AC002060, AC003075, AC006084, AC006084,AC006084, AC006144, AC006144, AC007000, AC007000, AC007000, AC007276,AC009501, AC009948, AC009948, AC009948, AL022069, AL022069, AL049875,AL078644, AL078644, AL110502, AL110502, AL133304, AC037472, AC037472,AC018568, AC018568, AC011432, AC011432, AC011432, AC021652, AC021652,AC021652, AC063920, AC063920, AC063920, AL133124, AL133124, AL133124,AP000974, AP000974, AC022194, AC022194, AC022194, AP001645, AC027552,AC027552, AC012036, AC012036, AC022845, AC022845, AC012447, AC012447,AC012207, AC012207, AC002379, AC002379, AC002379, AC006556, Z99758,AC007345, AC007345, AC010210, AC010210, AL356982, AC011153, AL355474,AC005023, AL031387, AL133370, AL133370, AL133370, AL354751, AL354751,AL354751, AC011123, AC011123, AC011123, AC018840, AC018840, AL355489,AL157899, AL157899, AP001562, AP001562, AP001562, AC025508, AC025508,AC011768, AC011768, AC005146, AC006984, AC006984, AC007358, AC011327,AC011327, AL031650, AL033403, AL033403, AL034408, AL034408, AC074245,AC074245, AC010798, AC020656, AL138682, AL138682, AC073118, AC073118,AC012600, AC012600, AC012600, AL138814, AL138814, AB045359, AC005090,AC005164, AC022078, AC022078, AF130343, AF130343, AL021408, AL033522,AL035689, AL035689, AL035689, AL035700, AL109628, AL157915, AL157915,AL157915, AL353812, AL354833, AL354833, AL355052, AL356802, AL365475,Z95400, AC010736, AC010736, AC023965, AC011401, AC011401, AL121933,AL121933, AL359754, AL359754, AL159175, AF027390, AC011823, AC011823,AC007849, AC007849, AL024507, AL024507, AL391071, AL391071, Z92542,Z92542, AL161645, AL161645, AL161645, AC022122, AC025555, AC006313,AC009289, AP000068, AL135978, AL132826, AL132826, AL158158, AL158158,AC021850, AL136327, AL136327, AC078802, AC078802, AC074250, AC068393,AL354708, AL354708, AL354708, AL359271, AL359271, AL356259, AC003099,AC005166, AC006195, AC007743, AP001821, AP000598, AP000598, AP001567,AP001567, AC023109, AL133385, AL139136, AC021201, AC007744, AL133381,AC015521, AC015521, AP001321, AP000409, AP000409, AC023555, AC008583,AC008583, AF001905, U69729, U69729, U69729, AC069525, AC069525, andAC015464. HSPAA60 39 526447 1-364 15-378 AI912648, AW387635, BF350647,Z98044, and Z98044. HFAEF57 40 534142 1-628 15-642 AV655597, AW967329,AW963498, AV706016, AW966767, and AL121984. HEGAH43 41 532596 1-42815-442 AA400429, AA994981, AA846419, AA453384, AI015471, AA992965,AA400538, AL360078, and AF327147. HAGDG59 42 534165 1-1720 15-1734AV694248, BE895909, BE903848, BG027942, AV651246, BG109867, BF240140,BF217526, BF669125, BE779936, AV650099, BF971092, AW875350, AW956342,BF107182, BF697022, BG166672, BF030619, BE881774, BE548671, BF247518,AI888053, BF667451, BE872808, AI768748, BF792803, N37046, N23484,BE872350, BF239058, AW664126, BF107464, W88681, AW338066, AW952476,AW402833, BE971415, AW853145, BF968304, AI636324, N24759, BF665132,BF213364, AA830565, AV697089, AA167203, AW023148, AI815125, AI685119,H98763, BE465545, AW853521, AW405572, AA481430, AW604402, AA481434,AA223067, AA902413, AW578436, BG258700, AI954984, AA045833, AI567716,BE856103, AA577610, H42133, AA553538, AA470843, AW467047, AW169016,AI961753, H38614, AA835545, AI262411, AW192401, AI193508, AA576473,AV660930, AW262909, AA263040, BE927225, AA481670, BE782154, W88620,AA394254, BG261374, AA730743, AA653560, R80477, AV651327, BF572366,AW193089, BF904780, AW470979, BF904779, AW295546, AA045967, AA329460,H42134, AA213836, R23904, BE243520, BE693582, AI263974, AW293723,AA481674, H61494, BF674778, R23903, R80672, BF032805, R61171, N79745,BF382094, H38856, AA328661, BF902314, BF243422, R27506, BF894060,T72506, AW361405, H62468, W07107, AI468319, AA362581, R27793, AW853797,AW337877, AW075817, BF031795, BE866675, BF905580, T82403, R27885,AW339053, AA377009, BF791479, AI708354, AA485696, BF239244, AW051074,BE866177, AI497897, AA485827, AW273624, BF905573, BF205089, AW022407,AI678575, Z21560, BF238897, BF699925, BE926278, BF239919, AW836245,AF126780, and Z64479. HNGBX63 43 532615 1-503 15-517 AL137077. HE2AG5044 532595 1-472 15-486 AA278487, AL357314, AL357314, AC009337, andAC026052. HCUIN80 45 534128 1-812 15-826 AW500684, AI755214, AA284247,AI754567, AA535216, AI754105, BF768846, AW576251, AI056177, BF725761,AA410788, AA714011, AW328331, AA485328, AI792521, AV710214, AA582073,AA714110, AA515048, BE138509, BE968744, AA651632, AV738383, AA502991,AI962030, AW023111, AA837686, AA515728, AI792499, AL079734, BE138594,AV756491, AU147162, AW575000, AI053688, BF675251, BF804385, AA503298,AW576326, BF915799, AL037052, N23913, BF725844, AA714288, BF939548,BE062159, AI053793, BF526964, AW069227, AI792575, AA084609, AW973992,AA809546, AL037771, BE066564, AA283730, AA312559, AA659832, AI345132,AI801505, AA176978, BF032064, AV762354, BE301584, BG222813, AI612142,AA630854, AV763026, AV763058, AV760915, AI923052, H07953, AI755038,AI669421, AV763057, AW970940, AW970571, AI742633, AV758067, AW969743,BE739230, AA701080, AW237905, T05118, BF854308, BE501593, AW798093,BF839876, AV655282, AA601278, AV702109, H58354, BF771774, BF527070,AA595661, AW962651, AA128511, AW819125, AA019973, AI754653, AW467607,AW965807, AA526326, BF916767, AA593537, AA523792, AV759199, AW468009,AW576034, AV742957, AI440117, AV760508, AA832145, BG250286, AA492584,N95817, BE896991, AA618316, AV763410, AV753973, AW816516, AA829036,AV653153, AI583252, BE877891, BG059972, BE049032, AV762633, BF681619,AI554471, AI311779, AA536040, T74524, AA526625, AC004020, AL121985,AC068999, AC007687, AL034548, AC005529, AC005034, AL353898, Z82205,AL355392, Z85996, AC011290, AC023051, AC000025, AC005527, AC003013,AF003528, AC022073, Z94056, Z84469, AC011495, AC006389, AC002300,AC009024, AF001549, AC006948, AC005971, AP000014, AC007934, AC012005,AC005823, AC007590, AL161731, AC006387, Z98304, AL445483, AC004832,AL035685, AC005627, AL358293, AC006460, AC003051, AL078581, AP001729,AL096800, AC010170, AL356240, AC005792, AC004831, AC006236, AF196970,AF168787, AP001436, AC034242, Z93023, AL356652, AC010305, AL121601,AL050317, AC007333, AC007358, AC010605, AP000156, AP000070, AC003956,AC007382, AL121891, AL121747, AC004629, AC002301, AC005207, AL137061,AC007251, L11910, AC008985, AC005740, AC018633, AC006538, AL078461,AC012309, AC007488, AC002310, AC002492, U73169, AC005338, AL022162,AC007221, AC006449, AC008379, AC002128, AC006028, Z84484, AL139099,AP000512, AL035467, AF001548, AC005324, Z83822, AC004103, AL031733,AC007387, AC006023, AF195953, U75931, AL139396, AC004156, AL117348,AB023049, AC002306, AF124731, AC009363, AL080242, AL049631, AL035587,AC008745, AC007160, AC006333, Z97196, AC011465, AC004686, AC002550,AL109976, AC007653, AC004804, AL162424, AC005048, AC002302, AC007690,AC002319, AL049712, AL139289, AC005726, AC002430, AL049634, AL137145,AL080243, AC004253, AL136137, AL136102, AP001725, AP000103, Z95114,AC006544, Z92844, AX000035, AC005081, AL035684, AB026899, AC007056,AC007384, AD000685, AC005821, AC003110, AC006120, AL096755, AP001753,AC012081, AC009333, AL035671, AL034420, AL008719, AC011452, AC011479,AD000684, AL031311, AC005751, AP001675, AC008266, AL049569, AL109805,AC008784, AL031005, Z68870, AC008812, AL450226, AF003627, AC002369,AC004025, AC004595, AL137039, AL121900, AC011445, AC005274, AC027312,AC008395, AL139384, AL138976, AC009516, AL035086, AL161941, AC009244,AL031289, AL137162, AL022328, AC007371, AC000029, AL117332, AL049697,J00083, AL139343, AC005220, AC002347, AC005952, AC004928, Z98752,AL031291, AC004796, Z96811, AP000553, AC009509, AC018712, AC003668,AC005369, AC020637, AL079304, AL118505, AC006511, AL163249, AC008928,AC002544, AC021016, AC007919, AL121572, AC020552, AL138741, AC022148,AC004089, AF001550, AL133545, AC005200, AL136304, AL050335, AL050349,AC006271, AF165926, AC003667, AL133174, AL133153, AC011469, AC006000,AC008403, AC008623, AC010677, AC022143, AL133382, AL135818, AL163279,and AC008465. HADCL29 46 532056 1-680 15-694 H03258, AI299317, N52545,AC025153, AC025153, and AC025153. HAPPS89 47 532135 1-842 15-856AI963720, AL046409, AV728425, AV710066, AF330238, AI270117, AV725423,AI334443, BF337291, AV733830, AV760042, AI431303, BF697673, BF241967,AI305766, BF677892, AI284640, AA490183, BF668217, AW193265, AI754658,AI613280, AV762098, BG249643, AV734666, BE276880, AV760777, AV764307,AI345654, AV762395, AV762535, AV759172, BF679304, AW963497, AW274349,AI350211, AV761362, AL138455, AW953071, AL044940, AL138265, AV763670,AV762064, BE049139, AV764241, AV740801, AA551503, AV764329, AV760057,BE276641, BF680074, AW438643, AW419262, AV763540, AW265385, AV763971,AI289067, AV761843, AW975425, AL042853, AF074677, AL037683, AL041690,AL039958, AI619997, AV761489, AW979060, AV760937, BG107801, AI688846,BE350475, AV761106, AI341664, AV759204, AW276827, AV764578, AW193432,AA244357, AI061334, AV718260, AL119691, AI754955, AV762505, AV760774,AV763354, AW965008, AV762645, AW303196, AI375710, AI281881, AV759437,AI133164, AV762959, AV729881, AV763449, AL045053, AU147104, AW083402,AW502975, AA483223, BG109996, AV759382, AW833862, BE150580, AW301350,AV763847, AI814735, AV760466, AA584201, BF216716, AW327868, BE895987,AL042420, AV725431, AW974109, AV702857, AV762139, AU151000, AV762111,AA491831, AV759505, BE253048, BF541120, AV732891, AI801482, BF854876,AI345681, BG222267, AI345675, BF827410, AI708009, BF311000, AA533333,AA610491, AV735370, AA587604, AA581903, BF965007, BF991286, AW406447,AV762050, BF939954, AI053672, AI064864, AI610920, AW975049, AI340453,AA470969, AV713243, BG059450, AA584167, AV759507, AI281903, BE967369,AV761745, AV718479, BF475381, AI344844, AA682912, AV763633, AL046205,AA631507, AV682003, AV719506, AA719805, BF679256, AV763255, AI744995,AV762015, AV735495, AV762067, AW407578, AI355206, BF684828, AV764530,AA649642, AA577906, AA623002, AV759274, AW503666, AI679782, AW500125,AV761786, AI307608, AW517388, AV658733, BF130605, AI732865, AW302013,AV742057, AI349850, AI559705, AW410400, AI076616, AV760039, AI307201,AV760106, AA664015, AW972312, AW517377, BF724372, AA680243, AW518220,BF679274, AW472872, BE139146, AW021583, BF681576, AV710774, AV759518,F36273, AW576391, BF691714, AW970896, AV763122, AV719422, AW083364,AV759329, AA720702, AV756693, AA829223, BG036665, BF793766, AI634384,AL041412, BG059314, AV760700, AV735239, AW408717, AI305547, AI471481,AV762009, AL120687, AW969629, AI799642, BF680041, H08791, AA525824,AW513362, AI133102, AW970848, AW995093, BF964720, AA491814, N53150,AP001718, AL137881, AC023275, AC007919, AP002026, AC008770, AL139388,AC005516, AP001675, AL096840, AL078590, AL117382, AL035411, AC004948,AC007404, AC005046, AP000501, AL157915, U18394, AF015149, AC002470,AL035665, AL445604, AC006989, U18391, AF302689, AL137141, AC004638,AL031650, U18395, X55925, AL353739, AC003080, AC004832, U57009,AC010088, X55926, AF265340, AL139009, AE000661, AC008279, AL353807,AC009087, AC004840, AC011495, AC008602, AC008372, AC006501, AL391867,AC025735, U85198, AL022302, X54180, AC010104, AC009244, AC008562,AC005666, AC005323, X54181, I51997, AL355497, AC006338, AF015148,AL138759, AL445223, AC007243, AF015151, AC083871, X54175, AC007620,AC006376, U95742, AC005393, AC004019, AC005019, U67221, AC022274,AL096776, X55931, AC007216, AL163853, AC004765, AF029308, AC007274,U18392, U02531, AC010879, AC007298, AC003003, AL161666, AP000855,AL360227, AL160237, U67231, AC005859, AC027319, X54176, AC006539,AC004987, AC008733, U63630, X54178, X53550, AC004816, AF217490,AF015156, AL354720, AC022013, AL133517, AC002400, AL135796, AC004848,Z93241, X55924, AC009223, AC009506, U18398, AC022027, AC006153,AL161892, AC004884, AC005244, Z99495, U18387, U57005, AC087093,AC068713, AF015147, AC002094, AC011310, AL096829, AC005699, U18396,AC020893, AP000548, AC005250, Z84814, U18399, AL033383, AL049762,AC006251, AF015157, AC006328, AL022323, AC004453, D83989, AL353696,AL121748, AC002377, U57006, AL135839, AL096863, AC023344, AC006925,U67233, U57008, Z49816, AC009470, AC010168, AC027689, AC005914,AC005923, AC006999, AL031660, AL020995, AL118520, AC004814, AC019187,AC027644, AF067844, AC009482, AC024952, AC007350, AC003977, AC006213,AC073893, AP001714, AL137228, AF010238, AC001231, AL078461, AL022163,AL356750, AL136418, AC020637, AC008534, AC004953, AL357141, AC002128,AC010726, AC021878, AC007364, AC010384, AL031432, AL359077, AL163247,AL158196, AC016995, AL161724, AC008249, AC022432, AL035683, AC010723,AC011749, AC004825, AL139352, X55927, AL109853, AC007011, AC004989,AL136372, U66059, X88791, X54179, AL138715, AP000117, AC003007,AL136081, AL355385, AL021406, AL390168, AC005988, AL035659, AL136173,AC018507, AC018758, AL121959, AL133480, AC009948, AL049874, AC006450,AL049868, AC009179, AL121751, AC010682, AC007363, Z98742, AL356288,AL050097, AC004808, AL035404, AC006983, AC005387, AC026398, AL157372,AP001687, AC005216, AC005257, U67801, AC004502, AC020873, AC020873,AC027442, AC022766, AL139131, AC073219, AL354696, AC023864, AC023864,AL355975, AC022795, AC068289, AL353659, AC010073, AL353577, AC015587,AC025975, AC072051, AC073849, AC025869, AC055861, AC010853, AC011036,AC016319, AC068519, AC027234, AC015604, AC021212, AC034243, AC068755,AL157775, AL356423, AC034137, AC068682, AC021164, AC009858, AC009858,AC067779, AC067779, AC024475, AC026936, AC073446, AC073446, AC041025,AC023309, AC067907, AP001992, AC016538, AC027584, AC027584, AC027408,AC027772, AC012461, AP001929, AC009095, AC009899, AC023672, AC055788,AL358492, AC024337, AL353139, AC060817, AC011233, AC019092, AC019092,AC022051, AC023968, AC021921, AC021901, AC068196, AC068196, AL161634,AL354657, AC073909, AC013758, AC021401, AL390917, AL390917, AC007459,AC002369, AC002369, AC036208, AC036208, AC009407, and AC009407. HFGAH4448 533132 1-1629 15-1643 BG169338, BG023827, BF525720, AW948966,AW968891, AA868896, AW043609, BE503306, BF671249, AA863046, AW051836,AI654795, AI808367, AW972853, AI745196, AA482288, AA421645, W84810,BF029772, AA525503, AA470086, AA946801, BE048921, R67044, AA495896,AA903005, AA482196, AA865021, AA425004, AI075164, AW275813, H23981,AI634761, W84811, R45305, AI378172, AI184932, H22701, BE935481,BE463530, AI613129, AW022383, H95118, H24272, AV710267, AW236030,AW444923, AI015394, R66160, BF512442, R20482, AI864299, AI783459,AI366098, AI696350, AI804999, AI628688, R32232, Z45604, Z41288,AI361717, BF372594, BF370363, BF372593, BE142713, BF370390, BF509363,AA398223, BG257307, AJ276674, AC023120, AC023120, and AC023120. HFIHZ9649 532046 1-695 15-709 AW079613. HFIUR10 50 532060 1-527 15-541BF195618, AA191239, AW969824, AA009856, AW019964, AA808036, BE677291,AW973259, AW023662, AV742957, AU146063, AI369580, BG109444, AU153717,AV709074, BG032605, AI357823, AW888719, AL110373, AI832009, AV708388,AV725797, BE150580, AA223512, AV734980, AA402529, AA595661, AW410201,AA683069, AA191418, AI144036, AW474168, BF681348, AI590458, AI590499,F08248, AW302048, AV760508, BE794962, AA665181, H07953, AW971071,AA654781, AV763410, AA749035, BF965290, AI609972, BF676985, AV708385,AW504485, AV762633, AW166808, AA282951, AI860535, AI792575, AA634889,AW302950, AL048060, AI254913, AW875172, AI281689, AA668587, AA084619,BF675051, AI354423, AA832077, AI733129, BF674550, AL041924, BE139451,H73550, AA828853, N39953, AW863393, AV757526, AI859946, AW976008,AW023111, AA747234, AI565084, AV710482, AW814024, AV710045, AW963482,AI355246, AA814925, BE077105, AA653182, AA664521, AW440305, AI054397,AA651639, BF725761, AV758073, H15652, BE280771, AW438542, T74524,AW191063, BF940118, AW968205, AV762973, AA552578, BF965924, BF879045,AI251034, AI251203, AI251284, AW805539, BG236628, BE878259, AI250552,AA632556, BF809041, BG029224, BF868994, AW020736, AF236698, BE139139,AW271904, BF978025, BF681424, AU118374, AV758790, BG110480, AI803809,AV758097, AA574442, AV733434, BE155302, AA644664, AI792521, BE246472,BE901278, AA626825, AI686913, AV706237, BE155299, AW302293, AV702609,AA533123, BE968477, AV738383, BF814446, AI891080, AA516190, AA533040,AI284543, BE273825, AW779609, BF525663, AI380617, BF914419, AL079734,BG166965, AW069227, AL043351, AI267161, AV762870, AV658819, AV709273,AL042735, AA503018, AI973173, AL046746, BE062357, AI963705, T69857,AV730245, BF810071, AW301736, Z97987, AC020913, AL031281, AC007637,AL133456, Z93017, AC087225, Z83840, AF245699, AC010349, AC004106,AC004132, AC008925, AC004990, AC010618, AC006275, AL035405, AC006930,AF156495, AP001732, AL139824, AC003037, AC005162, AL050341, AL034420,AC024075, AL117382, AC008521, AP001039, AC005778, AL049569, AL109914,Z95152, AL163541, AC006367, AC005684, AL117377, AL109828, AL031681,AC007488, AC007425, AC007934, AL078602, AC005038, AC009743, AC006538,AC053467, Z95115, AC010203, AC010150, AC006545, AC006546, AC004970,AP001696, AL390736, AC003035, AC007381, AC006253, AC022173, AC003684,AC009331, AL109823, AC004605, AL035682, AC006270, AC016526, AC003664,AL078634, AL009031, AL137802, AJ251973, AC002326, AL356421, AC006388,AL138721, AC004103, AL139150, AP001718, AL109915, AL117187, AC004616,AC017100, AC006080, AC005027, AC017006, AL049540, AC005522, AP000353,AC008008, AC078843, AL161657, AL031280, AL035696, Z81364, AL157938,AL389883, AL024474, AC008266, AC007784, AC002996, AL354816, AC011449,AP000193, AL022313, AC008379, AC005004, AC005514, AL118501, AL009028,Z86064, AL035249, AC002549, AL109984, AF001549, Z84467, AL034419,AC006204, AL353574, AC006960, AC009079, AC009503, D87009, AC005014,AC005859, AC003662, AL035089, AC005751, AL390738, AL133246, AP000694,AL133355, AC004854, AC006237, AK023233, Z85999, AL022323, AC003046,AL031730, AL160071, Z82206, AF029308, AC009600, AC005498, AL133342,AP000348, AC011465, AC004812, AC007221, AL022316, AL035072, Z97630,AC008551, AC007685, AC005034, AL391602, AC005220, AP000117, AL023883,AL109825, AL137782, AL096800, AF252279, AC005695, AC007620, AL022237,AC006481, AC018812, AL035420, AC004611, Z84487, AC018719, AC010163,AL133344, AC011290, AC007292, AC011895, U63721, AC002352, AC011444,AC005327, AC004595, AC002470, AL008732, AC006460, AL137119, AC005786,AC006211, AF134726, AC010205, U52112, AC007597, AC016026, AF186191,AC005215, AF155238, AL121756, AL137796, AP001597, AL136526, AC006084,AC013434, AC010596, AL138836, AC011450, AC000063, AL354984, AC007201,AC022436, AC005784, AC005479, AC009319, AC005324, AC010489, AL049570,AC006014, AP000335, AJ297357, AC007679, AC004583, AL157877, AL023513,AC006162, AC004381, AF205588, AK023462, Z85986, AP000503, AC004522,AC007773, AC005527, AL035684, AL031847, AC004686, AL137784, AC005320,AL031680, AF001548, AL031597, AL139102, AL138807, AC010126, AC003048,AC008015, Z98036, AB030001, AC004000, and AC002357. HLDNA86 51 5357301-706 15-720 BG034488, BG120997, BE910635, BF314688, BE794497, BE875593,BF340441, BF306972, AA205661, BF203164, BG027046, AA643840, BF525733,BF982559, BG030710, BF792430, BE387269, AI955852, AA146971, AA553820,AI869318, BE276651, AI832232, AW026472, BF195525, BF058723, BF204545,W72049, AW248507, AI765058, AI765242, AA034035, AI884693, AI127262,W79426, AW872762, AI376148, BE391657, AI765029, AI073710, N63475,AI277854, AI339947, AW024138, BF061732, AW439832, AI694928, AA708126,AA813521, AI147240, AW242703, AI123913, AI498928, BG222313, AI268993,BE313144, AW079287, AI301956, AI311112, AI304651, BE314135, BF435947,AI347608, AI075899, AW839917, H99183, R47883, AI560747, BF969005,BE669425, AI346935, AI808064, BF195494, W46277, N23743, N30038,AW136724, AI342962, AA781466, AI338271, AI090370, AA625788, H81310,AA836303, BF205034, AA709462, AA399114, BE262728, H94295, AW204374,BF315735, AA156864, AI969210, AA041385, AV683814, AA708782, AA704970,AI686517, AW968213, AI038344, AA468399, AI423158, AI597845, BG152852,BF304202, AA741397, AA770182, AI219428, AA298982, W94777, N35718,AI374736, AA868201, AA041191, AI497678, AA974145, AA127044, AA468439,AA478010, AA707176, N36015, AW243034, AI203479, H81366, AI341986,AA513969, AA025410, AW204258, BE266232, AA993215, N59833, AV708035,AA991336, AA704288, AA745913, BE018923, AI291572, AW800090, AW000874,AV687411, BE393296, AI379942, AA033915, R06215, BE260130, AW404352,BE645626, AA146970, F27351, AA156956, AI357603, H45882, BE295615,AA542901, AA564888, AI357554, AA970265, AA564958, AI761938, AA935742,F30350, BF081714, AI762149, AI869708, AI220792, BF889167, N24466,AA044694, AW188811, AI919010, R47882, BE833225, AW235465, AW090329,AW961121, BE140096, BF591489, AI956005, AA298368, AV687766, AA398056,AI867544, AA298370, AA296786, W76401, AA478165, AI749204, N40747,W79326, AV661716, AA296693, AA298843, BE928658, H94190, AI369692,AI365332, AA029123, N44247, AI566992, AA918262, AA846379, AI269986,BF311814, W95282, AI915437, N94773, AA298624, AA041425, AA297475,N32448, AA125766, AA041429, F30226, AV687884, BE090937, R36822,AA045652, AV687881, BF750936, AA205774, T24460, AA297517, and AV730348.HNGAN75 52 532613 1-965 15-979 AL353810. HCUIO20 53 534220 1-366 15-380HLTEF12 54 532354 1-2009 15-2023 AL516839, AL515540, AU124773, BE793826,BE799663, BE791935, AI928841, BG177521, AW005472, BG164346, BE794165,BE744549, AL516204, BE383125, AU117173, BE617751, BE906822, BE617248,AA573936, AW470072, BE646485, BE797812, BE909124, BE740662, AI829978,AI339290, BE741031, AL516852, AW166889, AL534961, AI192955, AW780355,AL517045, AW157313, BE894493, BE293842, AW629424, AW025010, AW070803,AI086145, BE512673, AI418163, AI859725, AU137251, BE255112, AI819465,AI816039, AL534736, AW574541, BG236033, AW005461, AL515541, AA595260,BF313175, AI189477, AW512956, AW769006, AW087972, BE279989, BE900347,AW129615, AI963813, AW675506, BG249791, AL516840, AI093998, AW945181,BF974259, AA599151, AA599393, AI080082, AL531455, AI628281, AI189835,AI284394, BG033989, AI128105, AA194432, BE793622, AA181002, AU155416,AA773073, AI625249, AW156965, AI886855, BF317270, AU159778, AW469006,AU151863, BE272720, AI285334, AA668589, BE205937, BE293376, AI554091,AA194582, AA428271, AA724027, BE778798, AA973557, AL120284, AA977213,BE549412, F28501, AI082349, W20116, AA132428, AA314969, AL516205,BG055939, AI754545, BE793245, AA970441, AI128575, BE739619, T08672,AW472905, BE741487, AI186425, AA884851, AI051160, AI183740, BE739157,AL517046, AI475120, AI470142, AA159395, AI446226, AI241304, AA559012,W86231, AA996169, BE265404, AW950509, AA760649, AA115570, AA159298,F22270, F27528, AU155699, AA679907, M86179, T86865, AW974231, H24001,AV749630, AA807991, AW009279, BG163486, AA505630, AI261447, T31711,H20295, C06410, AW574621, BG035018, AI000329, H09218, C05284, AI097554,AW248736, F33051, AA455991, AI654382, AA194706, H09161, AL516853,AA156018, AA132384, BF771643, AA588330, AI240896, AA338059, R98122,N88680, BG105261, AA346227, C03909, AA009594, F17542, AA371843,BF447041, F36262, AU142328, BE044395, F30521, T70010, AA658541,AU129291, AI694006, C03698, T69940, T85208, H80955, AA346119, BG165873,AA847638, AU131579, BE733937, AW404115, AU131993, W86258, BF313032,AA508663, AA311533, AW169592, F17173, AA157549, AW168710, AA854197,BF509014, AW028494, F17657, AA182856, AL516626, AW341596, H21252,AI909957, AA095968, BF025747, AW002276, R29462, AU150634, BE262250,F26847, BG166716, AA156483, AU139963, AU151428, AU126058, AU126364,BG170152, AA773009, BE780783, BE783844, AU137337, AA341400, BF312126,AU153296, BE280416, BE540123, BF315935, X69433, U52144, M86719, X69432,U51167, A74327, A74799, N67429, N90435, and AA115091. HCFBJ91 55 5319601-871 15-885 AI253987, AI800426, AL042667, AL042670, AI343986, AA515048,BF805088, H63660, BE301610, AU120416, AI697235, AW301950, M78021,AI719298, AI697239, AI697242, BF847612, AW975164, BF769926, AI889995,AW303142, AL036282, AA664126, AW243831, AW468372, AI797998, AI423018,AA744030, AW302081, AI802804, AI620992, AF001298, AC002365, AL135960,AJ131016, AL096840, AL353807, AC007731, AC005500, Z94802, AL022316,AC005231, L78833, AL136223, AC005102, AC005004, AL031311, AL023583,AC004560, AC004033, Z93023, AL031447, AC004471, Z84466, AC005512,AL121891, AC027319, AL136137, AC004650, AC007055, AC005383, AC002565,AC005837, AC009003, AC005548, AC004000, AC021999, AC009086, AL049781,AC005839, AC002425, AC020754, AC007899, AF129756, AC000075, AC011479,Z95115, AL121658, Z93015, AL133324, AL135978, AC009516, AL157877,AC007242, AL096791, AC005355, Z97876, AL031282, AC005484, AC008812,AF217413, AP000211, AP000133, AF207550, AC004799, AL353804, Z92542,AC009314, AL022323, AC009953, AC008848, AC013447, AL109802, AC008733,AC005666, AL049540, AL031075, AC009247, AC007216, AL121586, AC005914,AC005664, AC004087, AL110115, AP000279, AP001718, AL109798, AB015355,AF190464, AL133163, AC002477, AC005522, AC006084, AC002357, AP000512,L78810, AC004846, AL035400, AL049839, AL133507, U91321, AC009756,AC004826, AL049576, AC005103, AC011449, AL121748, AC005722, AL109804,AC002036, AC004448, AF047825, AL132642, AC021016, AF196969, AC004895,AL109806, AC008543, U95742, AC005527, AC002997, AP000500, AP000504,AL138807, AL359382, AC020916, AL008726, AC009477, AC008126, AP000295,AC009194, AC006547, AP000038, AP000106, AC007688, AD000092, AL161670,Z98200, AC006241, AC018720, AL031772, AP000348, AC004106, AC007404,X55448, AK024122, AC005052, AC004167, AC008616, AP000510, AC008623,AC006441, AL031584, AL137162, AC004526, AC008403, AL109935, AC005399,AC005049, AC005585, AC004967, AL353777, AC005229, AL035658, U52112,AL049757, AC002394, Z98941, AC008753, AC006285, AC011464, AC002126,AC012442, AL121890, AL050307, AC002091, AF305872, AL117348, U73634,AC005740, AC004812, AC002504, AL159977, AL022313, AC005020, AC006115,AC004686, AC005015, AL109840, AL035681, AC010422, AC007546, AC004832,AP000113, AP000045, AC000085, AL109743, AL031255, AP001711, AC005529,AC007739, AC005086, AC006023, AC007383, AC018644, AC008395, AL139353,AC007845, AC008745, AF196779, AF165926, AC009228, AC010326, AF215937,AC008392, AC004099, AL034379, AF038458, AC026729, AC007620, AC005094,AC011450, AC011455, AL035458, AC068999, AC007052, AL049539, AC022075,and AC022075. HHFHP90 56 534615 1-1092 15-1106 AW021674, AV759517,AL046620, AI547110, BE139230, AA640305, AV683406, AI254267, AA180056,AI344906, AI318548, AA425283, AA493464, AW839858, AI174703, BE244547,AA524604, AA046906, AW162314, AA280886, AW516250, AI572680, BF724416,AA157876, AI251024, AA631915, AA493245, AW962791, AA019793, AU146789,AI753131, BF837032, AI433952, AW975239, BG180320, AW963552, AW162332,AW020612, AA661583, AW265468, BE042324, AL121039, BE967607, AI702049,AA828840, BF725436, AW631267, BF679568, AA084439, AW473160, AW575808,H62123, BG223498, AI570067, AA804177, AA935827, AA101744, AI114543,AI819419, AI270280, AA994282, AI076729, AA629668, AV763460, AI890297,AW338376, AA171400, BG059139, AA218684, AA604601, AW410844, AI253347,AA313025, AI567676, AI028148, AA167656, AA404619, BE049409, AV755245,AI610814, AA632355, AW514844, AI828721, AI754926, AI457152, BG059924,AW084152, AV760048, AW148821, BE150831, BF681208, R92703, AA133568,AI281622, AA569220, AW029626, AA182928, BF884488, BE045167, AA593168,BG003974, AI469089, AW850985, AA362439, BF882222, AI744963, BF846619,AI888050, AU157188, AW963295, AI376687, AW962971, AV728973, AI921744,AV764119, AA578711, AA600127, AI884404, BF207643, AA664963, BE152006,AI434103, AA622637, AI520984, AW880986, AA084320, BF914416, BF790866,BF849260, AA298365, AI003068, BF739035, BG250794, AI064968, W03800,AW243817, AA515727, AI308529, AW974283, BF944618, AI889177, AU158814,BF904892, AI745666, BF882223, AA779599, N49298, AI744890, BE044000,AU120977, AI275631, AA554289, AW157128, AW771679, BF942991, AI860648,BF970107, AA315052, AA601336, BF880881, AA491941, AI307563, AA047057,N99245, AA746951, AW028376, AA776663, AA729004, BE156611, AW022796,AA018258, BE875478, N26159, AA179364, AA244267, AI890857, AI907506,AI274773, BE155101, AA618531, AA487053, H47461, AI300608, AC009032,AC004938, AL135818, AP001670, AC005839, AF200465, AC008518, AC002326,AC011462, AC004223, AL009031, AC005914, AL022318, AC002524, AC022316,AF205588, AC007686, AK024511, AP000553, AL096701, AL034423, AL035587,AC000353, AC007919, AL356747, AC005015, AC007051, AE111169, AP001052,AL022323, AC018738, AP001752, AC005377, AC006252, AC005516, AC011450,AP000963, AC008543, AC011529, AL135749, AP001716, AC005368, AC005911,AC008760, Z85987, AC007021, AC006942, U80017, AL117337, AL133332,AP000289, AC010877, AP000042, AP000110, AC016554, AC006120, AC004955,AL121580, AL136223, Z97056, AC006440, AL353804, L47222, AL022320,AL109797, AL133448, AC011480, AL031663, AC008403, AC009314, AC007722,AC006511, AC006211, AC002350, AC016995, AC006277, AF023268, AC008536,AC004999, AP000502, AP000365, AC007956, U89337, AP000547, AJ400877,AC005412, Z68756, AL157760, AL049547, AC011510, AL121586, AL136967,AL050331, AL365444, AC011559, AC010326, AC005301, AL031662, AC003074,AC003029, AC005581, AC008764, AC005000, AC003665, AC003037, AK026694,U52852, AC005484, AC005730, AL133163, AF168787, AC010527, AL031846,AL031680, AC010363, AC009003, AL049569, Z82244, AC007345, AL133355,AC006261, AL050349, AL020993, AC008264, AL157952, AC005066, AL096814,AC007878, AL008726, AD000864, AF067844, AC008812, AC007263, Z73979,AC011475, AC020626, AC002553, AC009756, AC009060, AC002477, AL035460,AC008070, AC005779, AC004006, AL353807, AL136992, AK026729, AC006312,AC009530, AC002126, AL033529, AC012309, AL109758, AL391995, AL357497,AP000031, AC005335, AL353653, AC005821, AL136228, AL033533, AP000039,U50871, AL136418, AC006204, AL139182, AL109935, AF317635, AL020997,AC005755, AL161937, AP000280, AL136295, AL133211, Z93931, AL133418,AF190464, Z86090, AC007240, AC005740, AC005049, AL136362, AL024498,AL022315, AL031281, AC009477, AP001727, AC002303, AL139100, AC004231,AC005618, AB035195, AC004783, AC007098, AP000107, AC004847, AP000343,Z93017, AC005084, AC006064, Z82184, AL031229, AC006449, AC004967,AC009455, AC006974, AC008651, AF165926, AL122001, AC009399, AL355476,AC021016, AC010480, AF228703, AC005840, AL031311, AJ003147, AC006969,AL050335, Z83840, AC002464, AC019155, AC006345, AC005358, AL136971,AC020552, AC006487, AL354696, AL022165, AP000337, AL035072, AC005726,AL078639, AX026165, AC005207, AC017092, AC004814, AP000694, AC011490,AC000075, AL031295, AL121601, AP000215, Z93020, AC002544, AC007216,Z81008, AC010328, AC073481, AP001362, AP001362, AC022488, AC022488,AC022488, AP000803, AP000803, AP001459, and AP001459. HLYCQ48 57 5277571-750 15-764 BE139139, AI250552, AI254770, AI284543, AI821273, AI251284,AI251034, AI251203, AI223626, BE138387, AW970571, AI249853, AW238341,AW303098, AI791718, BE042006, AA501867, AW890425, AA613630, BF805088,AA676592, AA682912, AW970987, AW276678, BF916055, AI973173, AA574000,H68343, BE139133, AV743776, BE676912, AL037929, AW302017, AW162288,AW270771, AL037932, AL037923, AV743864, AA873573, AA302661, AA572983,AW338506, AI343986, AW236219, AV762633, AA525293, AA503018, AL042667,AL042670, AW591754, BF764256, C06141, BF870951, AI431434, AB039887,AP001628, AP001748, AC007227, AL135927, AC005738, AL358777, AL021154,U78308, AC002310, AC010458, AC006483, AL022319, AF141309, AC005339,AL034420, AC025588, I34294, AC006511, AF031078, AC005261, AL109627,AC005840, AP001727, U95742, AL022322, AL137129, AC002316, AC007226,AC003109, AP000346, AL121900, Z83844, AP001753, AC024078, AC007057,AL049872, AL121952, AL035704, AC022515, AP000501, AC008760, AL035400,AF196969, AF030876, AC006487, AL121897, AL049829, AC018633, AC004491,AC005932, AC005863, AC009060, AL022320, AC006312, AL031602, AC008745,AL122015, AC006512, Z98304, AC011464, AL354864, AC004033, AL035464,AC011500, AC007249, AC005251, AF036581, AC007216, AC020916, AF196779,AC005920, AC005924, AL020995, AP000347, AC002375, AC004832, AC020947,Z92546, AC005081, AC005488, AC005837, AC005088, AC007653, AC007687,AC011290, AL049709, AC008821, AC008924, AL022311, AC007850, AL355916,AC007404, AF064090, AC006041, AF045555, U91326, AC006251, AC000025,AL033529, AC009228, AC008115, AL136304, AL024498, AL137073, AC005156,AC007845, AL034405, AC005527, AL353804, AC008543, AC008764, AC008403,AC007679, AL031672, AL121586, AF243527, AL121751, AF317635, AC004149,AL139331, AP000275, AC004953, AC018751, AL031007, AP000353, AL137918,AL109928, U47924, AL133174, AC016026, AL353653, AP000022, AC002544,AC004645, AP000553, AL008719, AL121899, AC011497, AC016025, AC009516,AC011479, AL109984, Z83733, AC002553, AP000105, AP000037, AC008392,AF038458, AC011551, AP000359, AC005102, AL158040, AC004883, AL022336,AC007051, AL161725, AC006241, AL139100, AP001426, AC008747, AL135744,Z83845, AL139392, AC011449, AC009086, AC007565, AL390026, Z82215,AC004025, AC007191, Z98749, AC005049, AC006257, AC005529, AL031280,AL022165, AC003030, AL049749, AB041992, AP000114, AP000046, AC004089,AP001752, AP001725, AC005071, AL138787, AC009517, AC007405, AL109921,AC005618, AC020898, AC008537, AL035683, AP000696, AC007731, AL034379,U07563, AC003041, AL358372, AL020993, AC008569, AL031848, AL022476,AC002559, AL157406, AC027319, AP001712, AC006111, AC011462, AC004222,AC005778, AL132777, AC005593, AL117381, AC005274, AL031284, AC021036,AL022163, AJ400877, AF207550, AL157838, and AP000350. HHLAB07 58 5326021-724 15-738 AA309074, and AC010764. HFOXE30 59 532120 1-427 15-441AF327179, AL031273, Z96270, AL031273, AL031273, AL031273, and AC074234.HBJEL68 60 531924 1-770 15-784 BG150876, AI741650, AL159973, andAL159973. HFOXA73 61 850699 1-526 15-540 AC005866, AC007618, andAC005866. HFIUR35 62 532062 1-590 15-604 AA127598, T77777, AC009778,AC063924, and AC063924. HFPDE86 63 527728 1-738 15-752 AA649642,AI963720, AA610491, AI431303, BF668217, AA507824, AI224093, AW504669,AI580652, BE742023, AV759204, BE160516, AW023672, AA226153, AA664581,AA806796, AW979060, AA601492, BF680041, AA523837, AA613227, BE677026,AV764329, AI589230, AL042420, AA503015, AW169537, AV691147, F25867,AV760543, AC007390, AC009060, AP000557, AC005531, AC016026, AL353807,AC011495, AC005229, AL354720, AC007957, AC011442, AL009181, AF001552,AF088219, AL356414, AC006483, AP000201, AC005081, AC009756, AP000097,AC010271, AC009244, AC007956, AP000240, AC004675, AP000500, AC007216,AC008760, AL121655, U95742, AC008764, AC023490, AL353748, Z93017,AC007226, AC003962, AL133448, AP000556, AL161670, AL109825, AC008569,AC005004, AP001705, AL034417, AC004812, AL033392, AC005258, AC010513,AC007676, Z83840, AC010289, AC018682, AC004166, AC004971, AC007999,AL023494, Z98744, AL050348, AC004965, AL352984, AC006011, AC010168,AC004895, AL117336, AL136137, AC005071, AC007055, AF243527, AL096712,AL133289, AL022318, AC005520, AP001331, AC006207, AP001708, AL023575,AC005015, AC006241, AL023879, AL136179, AC004089, AC027319, AC008518,AL109804, AC009228, AL359457, AP001748, AC016637, AL138787, AC003070,AP001629, AC007227, AC008687, U91322, AL137073, AP000140, AC002303,AC005730, AJ277557, AP001692, AC008753, AC010582, AC005776, AC009247,AL009179, AC020750, AC010150, AL157838, AC007619, AL132639, AL135927,U89336, AL121983, AC016627, AL157938, AF165926, AC005231, AC007537,AC007050, AL139023, AL049780, AL442166, AP000088, AC005011, AL031904,AC004887, Z93930, AC003109, AL163284, AJ009614, AC002425, AC009470,AP001759, AC009086, AL117186, AP001753, AC004922, Z98050, AC005355,AL034420, AC006571, AC006079, AL080242, AL163301, AC007390, AC007390,and AC007390.

TABLE 4 Code Description Tissue Organ Cell Line Disease Vector AR022a_Heart a_Heart AR023 a_Liver a_Liver AR024 a_mammary gland a_mammarygland AR025 a_Prostate a_Prostate AR026 a_small intestine a_smallintestine AR027 a_Stomach a_Stomach AR028 Blood B cells Blood B cellsAR029 Blood B cells activated Blood B cells activated AR030 Blood Bcells resting Blood B cells resting AR031 Blood T cells activated BloodT cells activated AR032 Blood T cells resting Blood T cells restingAR033 brain brain AR034 breast breast AR035 breast cancer breast cancerAR036 Cell Line CAOV3 Cell Line CAOV3 AR037 cell line PA-1 cell linePA-1 AR038 cell line transformed cell line transformed AR039 colon colonAR040 colon (9808co65R) colon (9808co65R) AR041 colon (9809co15) colon(9809co15) AR042 colon cancer colon cancer AR043 colon cancer coloncancer (9808co64R) (9808co64R) AR044 colon cancer 9809co14 colon cancer9809co14 AR045 corn clone 5 corn clone 5 AR046 corn clone 6 corn clone 6AR047 corn clone 2 corn clone 2 AR048 corn clone 3 corn clone 3 AR049Corn Clone 4 Corn Clone 4 AR050 Donor II B Cells 24 hrs Donor II B Cells24 hrs AR051 Donor II B Cells 72 hrs Donor II B Cells 72 hrs AR052 DonorII B-Cells 24 hrs. Donor II B-Cells 24 hrs. AR053 Donor II B-Cells 72hrs Donor II B-Cells 72 hrs AR054 Donor II Resting B Cells Donor IIResting B Cells AR055 Heart Heart AR056 Human Lung (clonetech) HumanLung (clonetech) AR057 Human Mammary Human Mammary (clontech) (clontech)AR058 Human Thymus Human Thymus (clonetech) (clonetech) AR059 Jurkat(unstimulated) Jurkat (unstimulated) AR060 Kidney Kidney AR061 LiverLiver AR062 Liver (Clontech) Liver (Clontech) AR063 Lymphocytes chronicLymphocytes chronic lymphocytic leukaemia lymphocytic leukaemia AR064Lymphocytes diffuse Lymphocytes diffuse large B cell lymphoma large Bcell lymphoma AR065 Lymphocytes follicular Lymphocytes lymphomafollicular lymphoma AR066 normal breast normal breast AR067 NormalOvarian Normal Ovarian (4004901) (4004901) AR068 Normal Ovary NormalOvary 9508G045 9508G045 AR069 Normal Ovary Normal Ovary 9701G2089701G208 AR070 Normal Ovary Normal Ovary 9806G005 9806G005 AR071 OvarianCancer Ovarian Cancer AR072 Ovarian Cancer Ovarian Cancer (9702G001)(9702G001) AR073 Ovarian Cancer Ovarian Cancer (9707G029) (9707G029)AR074 Ovarian Cancer Ovarian Cancer (9804G011) (9804G011) AR075 OvarianCancer Ovarian Cancer (9806G019) (9806G019) AR076 Ovarian Cancer OvarianCancer (9807G017) (9807G017) AR077 Ovarian Cancer Ovarian Cancer(9809G001) (9809G001) AR078 ovarian cancer 15799 ovarian cancer 15799AR079 Ovarian Cancer Ovarian Cancer 17717AID 17717AID AR080 OvarianCancer Ovarian Cancer 4004664B1 4004664B1 AR081 Ovarian Cancer OvarianCancer 4005315A1 4005315A1 AR082 ovarian cancer ovarian cancer 9412730394127303 AR083 Ovarian Cancer Ovarian Cancer 96069304 96069304 AR084Ovarian Cancer Ovarian Cancer 9707G029 9707G029 AR085 Ovarian CancerOvarian Cancer 9807G045 9807G045 AR086 ovarian cancer ovarian cancer9809G001 9809G001 AR087 Ovarian Cancer Ovarian Cancer 9905C032RC9905C032RC AR088 Ovarian cancer 9907 Ovarian cancer 9907 C00 3rd C00 3rdAR089 Prostate Prostate AR090 Prostate (clonetech) Prostate (clonetech)AR091 prostate cancer prostate cancer AR092 prostate cancer #15176prostate cancer #15176 AR093 prostate cancer #15509 prostate cancer#15509 AR094 prostate cancer #15673 prostate cancer #15673 AR095 SmallIntestine Small Intestine (Clontech) (Clontech) AR096 Spleen SpleenAR097 Thymus T cells activated Thymus T cells activated AR098 Thymus Tcells resting Thymus T cells resting AR099 Tonsil Tonsil AR100 Tonsilgeminal center Tonsil geminal center centroblast centroblast AR101Tonsil germinal center B Tonsil germinal cell center B cell AR102 Tonsillymph node Tonsil lymph node AR103 Tonsil memory B cell Tonsil memory Bcell AR104 Whole Brain Whole Brain AR105 Xenograft ES-2 Xenograft ES-2AR106 Xenograft SW626 Xenograft SW626 AR119 001: IL-2 001: IL-2 AR120001: IL-2.1 001: IL-2.1 AR121 001: IL-2_b 001: IL-2_b AR124 002:Monocytes 002: Monocytes untreated (1 hr) untreated (1 hr) AR125 002:Monocytes 002: Monocytes untreated (5 hrs) untreated (5 hrs) AR126 002:Control.1C 002: Control.1C AR127 002: 1L2.1C 002: 1L2.1C AR130 003:Placebo-treated 003: Placebo-treated Rat Lacrimal Gland Rat LacrimalGland AR131 003: Placebo-treated 003: Placebo-treated Rat SubmandibularRat Submandibular Gland Gland AR135 004: Monocytes 004: Monocytesuntreated (5 hrs) untreated (5 hrs) AR136 004: Monocytes 004: Monocytesuntreated 1 hr untreated 1 hr AR139 005: Placebo (48 hrs) 005: Placebo(48 hrs) AR140 006: pC4 (24 hrs) 006: pC4 (24 hrs) AR141 006: pC4 (48hrs) 006: pC4 (48 hrs) AR152 007: PHA (1 hr) 007: PHA (1 hr) AR153 007:PHA (6 HRS) 007: PHA (6 HRS) AR154 007: PMA (6 hrs) 007: PMA (6 hrs)AR155 008: 1449_#2 008: 1449_#2 AR161 01: A - max 24 01: A - max 24AR162 01: A - max 26 01: A - max 26 AR163 01: A - max 30 01: A - max 30AR164 01: B - max 24 01: B - max 24 AR165 01: B - max 26 01: B - max 26AR166 01: B - max 30 01: B - max 30 AR167 1449 Sample 1449 Sample AR1683T3P10 1.0 uM insulin 3T3P10 1.0 uM insulin AR169 3T3P10 10 nM Insulin3T3P10 10 nM Insulin AR170 3T3P10 10 uM insulin 3T3P10 10 uM insulinAR171 3T3P10 No Insulin 3T3P10 No Insulin AR172 3T3P4 3T3P4 AR173Adipose (41892) Adipose (41892) AR174 Adipose Diabetic Adipose Diabetic(41611) (41611) AR175 Adipose Diabetic Adipose Diabetic (41661) (41661)AR176 Adipose Diabetic Adipose Diabetic (41689) (41689) AR177 AdiposeDiabetic Adipose Diabetic (41706) (41706) AR178 Adipose Diabetic AdiposeDiabetic (42352) (42352) AR179 Adipose Diabetic Adipose Diabetic (42366)(42366) AR180 Adipose Diabetic Adipose Diabetic (42452) (42452) AR181Adipose Diabetic Adipose Diabetic (42491) (42491) AR182 Adipose Normal(41843) Adipose Normal (41843) AR183 Adipose Normal (41893) AdiposeNormal (41893) AR184 Adipose Normal (42452) Adipose Normal (42452) AR185Adrenal Gland Adrenal Gland AR186 Adrenal Gland + Whole Adrenal Gland +Brain Whole Brain AR187 B7 (1 hr) + (inverted) B7 (1 hr) + (inverted)AR188 Breast (18275A2B) Breast (18275A2B) AR189 Breast (4004199) Breast(4004199) AR190 Breast (4004399) Breast (4004399) AR191 Breast(4004943B7) Breast (4004943B7) AR192 Breast (4005570B1) Breast(4005570B1) AR193 Breast Cancer Breast Cancer (4004127A30) (4004127A30)AR194 Breast Cancer Breast Cancer (400443A21) (400443A21) AR195 BreastCancer Breast Cancer (4004643A2) (4004643A2) AR196 Breast Cancer BreastCancer (4004710A7) (4004710A7) AR197 Breast Cancer Breast Cancer(4004943A21) (4004943A21) AR198 Breast Cancer Breast Cancer (400553A2)(400553A2) AR199 Breast Cancer Breast Cancer (9805C046R) (9805C046R)AR200 Breast Cancer Breast Cancer (9806C012R) (9806C012R) AR201 BreastCancer (ODQ Breast Cancer (ODQ 45913) 45913) AR202 Breast Cancer BreastCancer (ODQ45913) (ODQ45913) AR203 Breast Cancer Breast Cancer(ODQ4591B) (ODQ4591B) AR204 Colon Cancer (15663) Colon Cancer (15663)AR205 Colon Cancer Colon Cancer (4005144A4) (4005144A4) AR206 ColonCancer Colon Cancer (4005413A4) (4005413A4) AR207 Colon Cancer ColonCancer (4005570B1) (4005570B1) AR208 Control RNA #1 Control RNA #1 AR209Control RNA #2 Control RNA #2 AR210 Cultured Preadipocyte Cultured(blue) Preadipocyte (blue) AR211 Cultured Preadipocyte Cultured (Red)Preadipocyte (Red) AR212 Donor II B-Cells 24 hrs Donor II B-Cells 24 hrsAR213 Donor II Resting B-Cells Donor II Resting B- Cells AR214 H114EP1210 nM H114EP12 10 nM Insulin Insulin AR215 H114EP12 (10 nM H114EP12 (10nM insulin) insulin) AR216 H114EP12 (2.6 ug/ul) H114EP12 (2.6 ug/ul)AR217 H114EP12 (3.6 ug/ul) H114EP12 (3.6 ug/ul) AR218 HUVEC #1 HUVEC #1AR219 HUVEC #2 HUVEC #2 AR221 L6 undiff. L6 undiff. AR222 L6Undifferentiated L6 Undifferentiated AR223 L6P8 + 10 nM Insulin L6P8 +10 nM Insulin AR224 L6P8 + HS L6P8 + HS AR225 L6P8 10 nM Insulin L6P8 10nM Insulin AR226 Liver (00-06-A007B) Liver (00-06-A007B) AR227 Liver(96-02-A075) Liver (96-02-A075) AR228 Liver (96-03-A144) Liver(96-03-A144) AR229 Liver (96-04-A138) Liver (96-04-A138) AR230 Liver(97-10-A074B) Liver (97-10-A074B) AR231 Liver (98-09-A242A) Liver(98-09-A242A) AR232 Liver Diabetic (1042) Liver Diabetic (1042) AR233Liver Diabetic (41616) Liver Diabetic (41616) AR234 Liver Diabetic(41955) Liver Diabetic (41955) AR235 Liver Diabetic (42352R) LiverDiabetic (42352R) AR236 Liver Diabetic (42366) Liver Diabetic (42366)AR237 Liver Diabetic (42483) Liver Diabetic (42483) AR238 Liver Diabetic(42491) Liver Diabetic (42491) AR239 Liver Diabetic (99-09- LiverDiabetic (99- A281A) 09-A281A) AR240 Lung Lung AR241 Lung (27270) Lung(27270) AR242 Lung (2727Q) Lung (2727Q) AR243 Lung Cancer Lung Cancer(4005116A1) (4005116A1) AR244 Lung Cancer Lung Cancer (4005121A5)(4005121A5) AR245 Lung Cancer Lung Cancer (4005121A5)) (4005121A5))AR246 Lung Cancer Lung Cancer (4005340A4) (4005340A4) AR247 MammaryGland Mammary Gland AR248 Monocyte (CT) Monocyte (CT) AR249 Monocyte(OCT) Monocyte (OCT) AR250 Monocytes (CT) Monocytes (CT) AR251 Monocytes(INFG 18 hr) Monocytes (INFG 18 hr) AR252 Monocytes (INFG 18 hr)Monocytes (INFG 18 hr) AR253 Monocytes (INFG 8-11) Monocytes (INFG 8-11)AR254 Monocytes (O CT) Monocytes (O CT) AR255 Muscle (91-01-A105) Muscle(91-01-A105) AR256 Muscle (92-04-A059) Muscle (92-04-A059) AR257 Muscle(97-11-A056d) Muscle (97-11- A056d) AR258 Muscle (99-06-A210A) Muscle(99-06- A210A) AR259 Muscle (99-07-A203B) Muscle (99-07- A203B) AR260Muscle (99-7-A203B) Muscle (99-7- A203B) AR261 Muscle Diabetic MuscleDiabetic (42352R) (42352R) AR262 Muscle Diabetic (42366) Muscle Diabetic(42366) AR263 NK-19 Control NK-19 Control AR264 NK-19 IL Treated 72 hrsNK-19 IL Treated 72 hrs AR265 NK-19 UK Treated 72 hrs. NK-19 UK Treated72 hrs. AR266 Omentum Normal (94- Omentum Normal 08-B009) (94-08-B009)AR267 Omentum Normal (97- Omentum Normal 01-A039A) (97-01-A039A) AR268Omentum Normal (97- Omentum Normal 04-A114C) (97-04-A114C) AR269 OmentumNormal (97- Omentum Normal 06-A117C) (97-06-A117C) AR270 Omentum Normal(97- Omentum Normal 09-B004C) (97-09-B004C) AR271 Ovarian Cancer OvarianCancer (17717AID) (17717AID) AR272 Ovarian Cancer Ovarian Cancer(9905C023RC) (9905C023RC) AR273 Ovarian Cancer Ovarian Cancer(9905C032RC) (9905C032RC) AR274 Ovary (9508G045) Ovary (9508G045) AR275Ovary (9701G208) Ovary (9701G208) AR276 Ovary 9806G005 Ovary 9806G005AR277 Pancreas Pancreas AR278 Placebo Placebo AR279 rIL2 Control rIL2Control AR280 RSS288L RSS288L AR281 RSS288LC RSS288LC AR282 SalivaryGland Salivary Gland AR283 Skeletal Muscle Skeletal Muscle AR284Skeletal Muscle (91-01- Skeletal Muscle (91- A105) 01-A105) AR285Skeletal Muscle (42180) Skeletal Muscle (42180) AR286 Skeletal Muscle(42386) Skeletal Muscle (42386) AR287 Skeletal Muscle (42461) SkeletalMuscle (42461) AR288 Skeletal Muscle (91-01- Skeletal Muscle (91- A105)01-A105) AR289 Skeletal Muscle (92-04- Skeletal Muscle (92- A059)04-A059) AR290 Skeletal Muscle (96-08- Skeletal Muscle (96- A171)08-A171) AR291 Skeletal Muscle (97-07- Skeletal Muscle (97- A190A)07-A190A) AR292 Skeletal Muscle Diabetic Skeletal Muscle (42352)Diabetic (42352) AR293 Skeletal Muscle Diabetic Skeletal Muscle (42366)Diabetic (42366) AR294 Skeletal Muscle Diabetic Skeletal Muscle (42395)Diabetic (42395) AR295 Skeletal Muscle Diabetic Skeletal Muscle (42483)Diabetic (42483) AR296 Skeletal Muscle Diabetic Skeletal Muscle (42491)Diabetic (42491) AR297 Skeletal Muscle Diabetic Skeletal Muscle 42352Diabetic 42352 AR298 Skeletal Musle (42461) Skeletal Musle (42461) AR299Small Intestine Small Intestine AR300 Stomach Stomach AR301 T-Cell +HDPBQ71.fc T-Cell + 1449 16 hrs HDPBQ71.fc 1449 16 hrs AR302 T-Cell +HDPBQ71.fc T-Cell + 1449 6 hrs HDPBQ71.fc 1449 6 hrs AR303 T-Cell + IL216 hrs T-Cell + IL2 16 hrs AR304 T-Cell + IL2 6 hrs T-Cell + IL2 6 hrsAR306 T-Cell Untreated 16 hrs T-Cell Untreated 16 hrs AR307 T-CellUntreated 6 hrs T-Cell Untreated 6 hrs AR308 T-Cells 24 hours T-Cells 24hours AR309 T-Cells 24 hrs T-Cells 24 hrs AR310 T-Cells 24 hrs. T-Cells24 hrs. AR311 T-Cells 24 hrs T-Cells 24 hrs AR312 T-Cells 4 days T-Cells4 days AR313 Thymus Thymus AR314 TRE TRE AR315 TREC TREC AR316 VirtualMixture Virtual Mixture H0009 Human Fetal Brain Uni-ZAP XR H0012 HumanFetal Kidney Human Fetal Kidney Kidney Uni-ZAP XR H0013 Human 8 WeekWhole Human 8 Week Old Embryo Uni-ZAP XR Embryo Embryo H0018 HumanGreater Human Greater peritoneum Uni-ZAP XR Omentum, fII remake OmentumH0024 Human Fetal Lung III Human Fetal Lung Lung Uni-ZAP XR H0026Namalwa Cells Namalwa B-Cell Lambda ZAP Line, EBV II immortalized H0031Human Placenta Human Placenta Placenta Uni-ZAP XR H0032 Human ProstateHuman Prostate Prostate Uni-ZAP XR H0036 Human Adult Small Human AdultSmall Small Int. Uni-ZAP XR Intestine Intestine H0038 Human Testes HumanTestes Testis Uni-ZAP XR H0041 Human Fetal Bone Human Fetal Bone BoneUni-ZAP XR H0046 Human Endometrial Human Endometrial Uterus diseaseUni-ZAP XR Tumor Tumor H0048 Human Pineal Gland Human Pineal GlandUni-ZAP XR H0050 Human Fetal Heart Human Fetal Heart Heart Uni-ZAP XRH0051 Human Hippocampus Human Hippocampus Brain Uni-ZAP XR H0052 HumanCerebellum Human Cerebellum Brain Uni-ZAP XR H0081 Human FetalEpithelium Human Fetal Skin Skin Uni-ZAP XR (Skin) H0083 HUMAN JURKATJurkat Cells Uni-ZAP XR MEMBRANE BOUND POLYSOMES H0087 Human ThymusHuman Thymus pBluescript H0090 Human T-Cell T-Cell Lymphoma T-Celldisease Uni-ZAP XR Lymphoma H0095 Human Greater Human Greater peritoneumUni-ZAP XR Omentum, RNA Omentum Remake H0099 Human Lung Cancer, HumanLung Cancer Lung pBluescript subtracted H0100 Human Whole Six Week HumanWhole Six Embryo Uni-ZAP XR Old Embryo Week Old Embryo H0111 HumanPlacenta, Human Placenta Placenta pBluescript subtracted H0123 HumanFetal Dura Mater Human Fetal Dura Brain Uni-ZAP XR Mater H0124 HumanHuman Sk Muscle disease Uni-ZAP XR Rhabdomyosarcoma RhabdomyosarcomaH0134 Raji Cells, Cyclohexamide Blood Cell Line Uni-ZAP XR cyclohexamidetreated Treated Cem, Jurkat, Raji, and Supt H0135 Human Synovial HumanSynovial Synovium Uni-ZAP XR Sarcoma Sarcoma H0136 Supt Cells,Cyclohexamide Blood Cell Line Uni-ZAP XR cyclohexamide treated TreatedCem, Jurkat, Raji, and Supt H0144 Nine Week Old Early 9 Wk Old EarlyEmbryo Uni-ZAP XR Stage Human Stage Human H0149 7 Week Old Early StageHuman Whole 7 Embryo Uni-ZAP XR Human, subtracted Week Old Embryo H0154Human Fibrosarcoma Human Skin Skin disease Uni-ZAP XR Fibrosarcoma H0156Human Adrenal Gland Human Adrenal Adrenal disease Uni-ZAP XR Tumor GlandTumor Gland H0163 Human Synovium Human Synovium Synovium Uni-ZAP XRH0165 Human Prostate Cancer, Human Prostate Prostate disease Uni-ZAP XRStage B2 Cancer, stage B2 H0169 Human Prostate Cancer, Human ProstateProstate disease Uni-ZAP XR Stage C fraction Cancer, stage C H0170 12Week Old Early Twelve Week Old Embryo Uni-ZAP XR Stage Human Early StageHuman H0171 12 Week Old Early Twelve Week Old Embryo Uni-ZAP XR StageHuman, II Early Stage Human H0172 Human Fetal Brain, Human Fetal BrainBrain Lambda ZAP random primed II H0178 Human Fetal Brain Human FetalBrain Brain Uni-ZAP XR H0181 Human Primary Breast Human Primary Breastdisease Uni-ZAP XR Cancer Breast Cancer H0196 Human Human Heart Uni-ZAPXR Cardiomyopathy, Cardiomyopathy subtracted H0199 Human Fetal Liver,Human Fetal Liver Liver Uni-ZAP XR subtracted, neg clone H0208 EarlyStage Human Human Fetal Lung Lung pBluescript Lung, subtracted H0211Human Human Prostate Prostate pBluescript Prostate, differentialexpression H0213 Human Pituitary, Human Pituitary Uni-ZAP XR subtractedH0216 Supt cells, Cyclohexamide Blood Cell Line pBluescriptcyclohexamide treated, Treated Cem, Jurkat, subtracted Raji, and SuptH0228 C7MCF7 cell line, C7MCF7 Cell Line, Breast Cell Line Uni-ZAP XRestrogen treated estrogen treated H0239 Human Kidney Tumor Human KidneyKidney disease Uni-ZAP XR Tumor H0254 Breast Lymph node Breast LymphNode Lymph Uni-ZAP XR cDNA library Node H0255 breast lymph node BreastLymph Node Lymph Lambda ZAP CDNA library Node II H0264 human tonsilsHuman Tonsil Tonsil Uni-ZAP XR H0265 Activated T-Cell T-Cells Blood CellLine Uni-ZAP XR (12 hs)/Thiouridine labelledEco H0266 HumanMicrovascular HMEC Vein Cell Line Lambda ZAP Endothelial Cells, fract. AII H0271 Human Neutrophil, Human Neutrophil - Blood Cell Line Uni-ZAP XRActivated Activated H0272 HUMAN TONSILS, Human Tonsil Tonsil Uni-ZAP XRFRACTION 2 H0288 Human OB HOS control Human Bone Cell Line Uni-ZAP XRfraction I Osteoblastoma HOS cell line H0294 Amniotic Cells - TNFAmniotic Cells - Placenta Cell Line Uni-ZAP XR induced TNF induced H0295Amniotic Cells - Amniotic Cells - Placenta Cell Line Uni-ZAP XR PrimaryCulture Primary Culture H0305 CD34 positive cells CD34 Positive CellsCord ZAP Express (Cord Blood) Blood H0306 CD34 depleted Buffy CD34Depleted Cord ZAP Express Coat (Cord Blood) Buffy Coat (Cord BloodBlood) H0309 Human Chronic Synovium, Chronic Synovium disease Uni-ZAP XRSynovitis Synovitis/ Osteoarthritis H0316 HUMAN STOMACH Human StomachStomach Uni-ZAP XR H0318 HUMAN B CELL Human B Cell Lymph disease Uni-ZAPXR LYMPHOMA Lymphoma Node H0328 human ovarian cancer Ovarian CancerOvary disease Uni-ZAP XR H0333 Hemangiopericytoma HemangiopericytomaBlood disease Lambda ZAP vessel II H0341 Bone Marrow Cell Line BoneMarrow Cell Bone Cell Line Uni-ZAP XR (RS4; 11) Line RS4; 11 MarrowH0351 Glioblastoma Glioblastoma Brain disease Uni-ZAP XR H0355 HumanLiver Human Liver, normal pCMVSport 1 Adult H0370 H. Lymph node breastLymph node with disease Uni-ZAP XR Cancer Met. Breast Cancer H0373 HumanHeart Human Adult Heart Heart pCMVSport 1 H0393 Fetal Liver, subtractionHuman Fetal Liver Liver pBluescript II H0402 CD34 depleted Buffy CD34Depleted Cord ZAP Express Coat (Cord Blood), re- Buffy Coat (Cord Bloodexcision Blood) H0422 T-Cell PHA 16 hrs T-Cells Blood Cell Line pSport1H0423 T-Cell PHA 24 hrs T-Cells Blood Cell Line pSport1 H0424 HumanPituitary, subt Human Pituitary pBluescript IX H0427 Human Adipose HumanAdipose, left pSport1 hiplipoma H0431 H. Kidney Medulla, re- Kidneymedulla Kidney pBluescript excision H0435 Ovarian Tumor 10-3-95 OvarianTumor, Ovary pCMVSport OV350721 2.0 H0436 Resting T-Cell Library, IIT-Cells Blood Cell Line pSport1 H0441 H. Kidney Cortex, Kidney cortexKidney pBluescript subtracted H0445 Spleen, Chronic Human Spleen, CLLSpleen disease pSport1 lymphocytic leukemia H0478 Salivary Gland, Lib 2Human Salivary Salivary pSport1 Gland gland H0483 Breast Cancer cellline, Breast Cancer Cell pSport1 MDA 36 line, MDA 36 H0484 Breast CancerCell line, Breast Cancer Cell pSport1 angiogenic line, Angiogenic, 36T3H0486 Hodgkin's Lymphoma II Hodgkin's disease pCMVSport Lymphoma II 2.0H0494 Keratinocyte Keratinocyte pCMVSport 2.0 H0497 HEL cell line HELcell line HEL pSport1 92.1.7 H0506 Ulcerative Colitis Colon ColonpSport1 H0509 Liver, Hepatoma Human Liver, Liver disease pCMVSportHepatoma, patient 8 3.0 H0510 Human Liver, normal Human Liver, LiverpCMVSport normal, Patient # 8 3.0 H0518 pBMC stimulated w/ pBMCstimulated pCMVSport poly I/C with poly I/C 3.0 H0520 NTERA2 + retinoicNTERA2, pSport1 acid, 14 days Teratocarcinoma cell line H0521 PrimaryDendritic Cells, Primary Dendritic pCMVSport lib 1 cells 3.0 H0522Primary Dendritic Primary Dendritic pCMVSport cells, frac 2 cells 3.0H0529 Myoloid Progenitor Cell TF-1 Cell Line; pCMVSport Line Myoloidprogenitor 3.0 cell line H0530 Human Dermal Human Dermal pSport1Endothelial Endothelial Cells; Cells, untreated untreated H0542 T Cellhelper I Helper T cell pCMVSport 3.0 H0543 T cell helper II Helper Tcell pCMVSport 3.0 H0544 Human endometrial Human endometrial pCMVSportstromal cells stromal cells 3.0 H0545 Human endometrial Humanendometrial pCMVSport stromal cells-treated stromal cells-treated 3.0with progesterone with proge H0547 NTERA2 NTERA2, pSport1teratocarcinoma cell Teratocarcinoma cell line + retinoic acid (14 linedays) H0549 H. Epididiymus, caput & Human Epididiymus, Uni-ZAP XR corpuscaput and corpus H0550 H. Epididiymus, cauda Human Epididiymus, Uni-ZAPXR cauda H0551 Human Thymus Stromal Human Thymus pCMVSport Cells StromalCells 3.0 H0553 Human Placenta Human Placenta pCMVSport 3.0 H0555Rejected Kidney, lib 4 Human Rejected Kidney disease pCMVSport Kidney3.0 H0556 Activated T- T-Cells Blood Cell Line Uni-ZAP XR cell(12h)/Thiouridine-re- excision H0560 KMH2 KMH2 pCMVSport 3.0 H0561 L428L428 pCMVSport 3.0 H0562 Human Fetal Brain, Human Fetal Brain pCMVSportnormalized c5-11-26 2.0 H0571 Human Fetal Brain, Human Fetal BrainpCMVSport normalized C500HE 2.0 H0574 Hepatocellular Tumor;Hepatocellular Liver disease Lambda ZAP re-excision Tumor II H0575 HumanAdult Human Adult Lung Uni-ZAP XR Pulmonary; re-excision Pulmonary H0580Dendritic cells, pooled Pooled dendritic cells pCMVSport 3.0 H0581 HumanBone Marrow, Human Bone Marrow Bone pCMVSport treated Marrow 3.0 H0586Healing groin wound, healing groin wound, groin disease pCMVSport 6.5hours post incision 6.5 hours post 3.0 incision - 2/ H0587 Healing groinwound; Groin-Feb. 19, 1997 groin disease pCMVSport 7.5 hours postincision 3.0 H0590 Human adult small Human Adult Small Small Int.Uni-ZAP XR intestine, re-excision Intestine H0593 Olfactory Olfactoryepithelium pCMVSport epithelium; nasalcavity from roof of left 3.0 nasalcacit H0597 Human Colon; re- Human Colon Lambda ZAP excision II H0599Human Adult Heart; re- Human Adult Heart Heart Uni-ZAP XR excision H0604Human Pituitary, re- Human Pituitary pBluescript excision H0606 HumanPrimary Breast Human Primary Breast disease Uni-ZAP XR Cancer;re-excision Breast Cancer H0615 Human Ovarian Cancer Ovarian CancerOvary disease Uni-ZAP XR Reexcision H0616 Human Testes, Human TestesTestis Uni-ZAP XR Reexcision H0617 Human Primary Breast Human PrimaryBreast disease Uni-ZAP XR Cancer Reexcision Breast Cancer H0618 HumanAdult Testes, Human Adult Testis Testis Uni-ZAP XR Large Inserts,Reexcision H0619 Fetal Heart Human Fetal Heart Heart Uni-ZAP XR H0620Human Fetal Kidney; Human Fetal Kidney Kidney Uni-ZAP XR ReexcisionH0622 Human Pancreas Tumor; Human Pancreas Pancreas disease Uni-ZAP XRReexcision Tumor H0624 12 Week Early Stage Twelve Week Old EmbryoUni-ZAP XR Human II; Reexcision Early Stage Human H0626 Saos2 Cells;Untreated Saos2 Cell Line; pSport1 Untreated H0632 HepatocellularHepatocellular Liver Lambda ZAP Tumor; re-excision Tumor II H0634 HumanTestes Tumor, Human Testes Tumor Testis disease Uni-ZAP XR re-excisionH0637 Dendritic Cells From Dentritic cells from pSport1 CD34 Cells CD34cells H0638 CD40 activated CD40 activated pSport1 monocyte dendridiccells monocyte dendridic cells H0641 LPS activated derived LPS activatedpSport1 dendritic cells monocyte derived dendritic cells H0643 Hep G2Cells, PCR Hep G2 Cells Other library H0644 Human Placenta (re- HumanPlacenta Placenta Uni-ZAP XR excision) H0645 Fetal Heart, re-excisionHuman Fetal Heart Heart Uni-ZAP XR H0646 Lung, Cancer (4005313Metastatic squamous pSport1 A3): Invasive Poorly cell lung carcinoma,Differentiated Lung poorly di Adenocarcinoma, H0647 Lung, Cancer(4005163 Invasive poorly disease pSport1 B7): Invasive, Poorlydifferentiated lung Diff. Adenocarcinoma, adenocarcinoma MetastaticH0648 Ovary, Cancer: Papillary Cstic disease pSport1 (4004562 B6)Papillary neoplasm of low Serous Cystic Neoplasm, malignant potentia LowMalignant Pot H0650 B-Cells B-Cells pCMVSport 3.0 H0651 Ovary, Normal:Normal Ovary pSport1 (9805C040R) H0652 Lung, Normal: (4005313 NormalLung pSport1 B1) H0653 Stromal Cells Stromal Cells pSport1 H0654 Lung,Cancer: (4005313 Metastatic Squamous Other A3) Invasive Poorly- celllung Carcinoma differentiated Metastatic poorly dif lung adenoc H0656B-cells (unstimulated) B-cells pSport1 (unstimulated) H0657 B-cells(stimulated) B-cells (stimulated) pSport1 H0658 Ovary, Cancer9809C332-Poorly Ovary & disease pSport1 (9809C332): Poorly differentiateFallopian differentiated Tubes adenocarcinoma H0659 Ovary, Cancer GradeII Papillary Ovary disease pSport1 (15395A1F): Grade II Carcinoma, OvaryPapillary Carcinoma H0660 Ovary, Cancer: Poorly differentiated diseasepSport1 (15799A1F) Poorly carcinoma, ovary differentiated carcinomaH0661 Breast, Cancer: Breast cancer disease pSport1 (4004943 A5) H0664Breast, Cancer: Breast Cancer Breast disease pSport1 (9806C012R) H0670Ovary, Cancer (4004650 Ovarian Cancer - pSport1 A3): Well- 4004650A3Differentiated Micropapillary Serous Carcinoma H0672 Ovary, Cancer:Ovarian Ovary pSport1 (4004576 A8) Cancer (4004576A8) H0673 HumanProstate Cancer, Human Prostate Prostate Uni-ZAP XR Stage B2;re-excision Cancer, stage B2 H0675 Colon, Cancer: Colon Cancer pCMVSport(9808C064R) 9808C064R 3.0 H0676 Colon, Cancer: Colon Cancer pCMVSport(9808C064R)-total RNA 9808C064R 3.0 H0684 Serous Papillary OvarianCancer- Ovaries pCMVSport Adenocarcinoma 9810G606 3.0 H0686Adenocarcinoma of Adenocarcinoma of pCMVSport Ovary, Human Cell LineOvary, Human Cell 3.0 Line, # SW-626 H0687 Human normal Human normalOvary pCMVSport ovary (#9610G215) ovary (#9610G215) 3.0 H0690 OvarianCancer, # Ovarian Cancer, pCMVSport 9702G001 #9702G001 3.0 H0695mononucleocytes from mononucleocytes pCMVSport patient from patient atShady 3.0 Grove Hospit N0004 Human Hippocampus Human Hippocampus S0003Human Osteoclastoma Osteoclastoma bone disease Uni-ZAP XR S0010 HumanAmygdala Amygdala Uni-ZAP XR S0011 STROMAL - Osteoclastoma bone diseaseUni-ZAP XR OSTEOCLASTOMA S0013 Prostate Prostate prostate Uni-ZAP XRS0022 Human Osteoclastoma Osteoclastoma Uni-ZAP XR Stromal Cells -Stromal Cells unamplified S0026 Stromal cell TF274 stromal cell BoneCell Line Uni-ZAP XR marrow S0027 Smooth muscle, serum Smooth musclePulmanary Cell Line Uni-ZAP XR treated artery S0028 Smooth muscle,control Smooth muscle Pulmanary Cell Line Uni-ZAP XR artery S0029 brainstem Brain stem brain Uni-ZAP XR S0031 Spinal cord Spinal cord spinalUni-ZAP XR cord S0038 Human Whole Brain #2 - Human Whole Brain ZAPExpress Oligo dT > 1.5 Kb #2 S0040 Adipocytes Human Adipocytes Uni-ZAPXR from Osteoclastoma S0044 Prostate BPH prostate BPH Prostate diseaseUni-ZAP XR S0045 Endothelial cells-control Endothelial cell endothelialCell Line Uni-ZAP XR cell-lung S0046 Endothelial-induced Endothelialcell endothelial Cell Line Uni-ZAP XR cell-lung S0049 Human Brain,Striatum Human Brain, Uni-ZAP XR Striatum S0051 Human Human diseaseUni-ZAP XR Hypothalmus, Schizophrenia Hypothalamus, Schizophrenia S0052neutrophils control human neutrophils blood Cell Line Uni-ZAP XR S0112Hypothalamus Brain Uni-ZAP XR S0114 Anergic T-cell Anergic T-cell CellLine Uni-ZAP XR S0116 Bone marrow Bone marrow Bone Uni-ZAP XR marrowS0126 Osteoblasts Osteoblasts Knee Cell Line Uni-ZAP XR S0142Macrophage-oxLDL macrophage-oxidized blood Cell Line Uni-ZAP XR LDLtreated S0144 Macrophage (GM-CSF Macrophage (GM- Uni-ZAP XR treated) CSFtreated) S0152 PC3 Prostate cell line PC3 prostate cell line Uni-ZAP XRS0180 Bone Marrow Stroma, Bone Marrow disease Uni-ZAP XR TNF&LPS indStroma, TNF & LPS induced S0192 Synovial Fibroblasts SynovialFibroblasts pSport1 (control) S0194 Synovial hypoxia SynovialFibroblasts pSport1 S0196 Synovial IL-1/TNF Synovial Fibroblasts pSport1stimulated S0206 Smooth Muscle- Smooth muscle Pulmanary Cell LinepBluescript HASTE normalized artery S0212 Bone Marrow Stromal BoneMarrow pSport1 Cell, untreated Stromal Cell, untreated S0218 ApoptoticT-cell, re- apoptotic cells Cell Line Uni-ZAP XR excision S0222 H.Frontal H. Brain, Frontal Brain disease Uni-ZAP XR cortex, epileptic;re- Cortex, Epileptic excision S0260 Spinal Cord, re-excision Spinalcord spinal Uni-ZAP XR cord S0276 Synovial hypoxia-RSF Synovialfobroblasts Synovial pSport1 subtracted (rheumatoid) tissue S0278 HMacrophage (GM- Macrophage (GM- Uni-ZAP XR CSF treated), re-excision CSFtreated) S0280 Human Adipose Tissue, Human Adipose Uni-ZAP XRre-excision Tissue S0298 Bone marrow Bone marrow Bone pSport1 stroma,treated stroma, treatedSB marrow S0312 Human Human osteoarthriticdisease pSport1 osteoarthritic; fraction II cartilage S0314 Human Humanosteoarthritic disease pSport1 osteoarthritis; fraction I cartilageS0330 Palate normal Palate normal Uvula pSport1 S0344 Macrophage-oxLDL;re- macrophage-oxidized blood Cell Line Uni-ZAP XR excision LDL treatedS0346 Human Amygdala; re- Amygdala Uni-ZAP XR excision S0354 ColonNormal II Colon Normal Colon pSport1 S0356 Colon Carcinoma ColonCarcinoma Colon disease pSport1 S0358 Colon Normal III Colon NormalColon pSport1 S0360 Colon Tumor II Colon Tumor Colon disease pSport1S0362 Human Gastrocnemius Gastrocnemius pSport1 muscle S0364 HumanQuadriceps Quadriceps muscle pSport1 S0366 Human Soleus Soleus MusclepSport1 S0374 Normal colon Normal colon pSport1 S0376 Colon Tumor ColonTumor disease pSport1 S0378 Pancreas normal PCA4 Pancreas Normal pSport1No PCA4 No S0380 Pancreas Tumor PCA4 Pancreas Tumor disease pSport1 TuPCA4 Tu S0390 Smooth muscle, control; Smooth muscle Pulmanary Cell LineUni-ZAP XR re-excision artery S0406 Rectum tumor Rectum tumor pSport1S0408 Colon, normal Colon, normal pSport1 S0410 Colon, tumor Colon,tumor pSport1 S0418 CHME Cell Line; treated CHME Cell Line; pCMVSport 5hrs treated 3.0 S0420 CHME Cell CHME Cell line, pSport1 Line, untreateduntreatetd S0422 Mo7e Cell Line GM- Mo7e Cell Line GM- pCMVSport CSFtreated (1 ng/ml) CSF treated (1 ng/ml) 3.0 S0426 Monocyte activated;re- Monocyte-activated blood Cell Line Uni-ZAP XR excision S0428Neutrophils control; re- human neutrophils blood Cell Line Uni-ZAP XRexcision S0434 Stomach Normal Stomach Normal disease pSport1 S0436Stomach Tumor Stomach Tumor disease pSport1 S0438 Liver Normal Met5NoLiver Normal pSport1 Met5No S0440 Liver Tumor Met 5 Tu Liver TumorpSport1 S0442 Colon Normal Colon Normal pSport1 S0444 Colon Tumor ColonTumor disease pSport1 S0450 Larynx Tumor Larynx Tumor pSport1 S0462Thyroid Thyroiditis Thyroid Thyroiditis pSport1 S0474 Human bloodplatelets Platelets Blood Other platelets S3012 Smooth Muscle SerumSmooth muscle Pulmanary Cell Line pBluescript Treated, Norm artery S3014Smooth muscle, serum Smooth muscle Pulmanary Cell Line pBluescriptinduced, re-exc artery S6024 Alzheimers, spongy Alzheimer's/Spongy Braindisease Uni-ZAP XR change change S6028 Human Manic Human Manic Braindisease Uni-ZAP XR Depression Tissue depression tissue T0006 HumanPineal Gland Human Pinneal pBluescript Gland SK- T0010 Human InfantBrain Human Infant Brain Other T0039 HSA 172 Cells Human HSA 172 cellpBluescript line SK- T0040 HSC172 Cells SA172 Cells pBluescript SK-T0041 Jurkat T-cell G1 phase Jurkat T-cell pBluescript SK- T0049 Aortaendothelial cells + Aorta endothelial pBluescript TNF-a cells SK- T0067Human Thyroid Human Thyroid pBluescript SK- T0091 Liver, hepatocellularpBluescript carcinoma SK- T0109 Human (HCC) cell line pBluescript liver(mouse) metastasis, SK- remake L0163 Human heart cDNA heart (YNakamura)L0354 JG, Human foetal Bluescript Kidney tissue L0355 P, Human foetalBrain Bluescript Whole tissue L0361 Stratagene ovary ovary Bluescript SK(#937217) L0362 Stratagene ovarian Bluescript cancer (#937219) SK- L0367NCI_CGAP_Sch1 Schwannoma tumor Bluescript SK- L0370 Johnston frontalcortex pooled frontal lobe brain Bluescript SK- L0371 NCI_CGAP_Br3breast tumor breast Bluescript SK- L0372 NCI_CGAP_Co12 colon tumor colonBluescript SK- L0373 NCI_CGAP_Co11 tumor colon Bluescript SK- L0374NCI_CGAP_Co2 tumor colon Bluescript SK- L0375 NCI_CGAP_Kid6 kidney tumorkidney Bluescript SK- L0384 NCI_CGAP_Pr23 prostate tumor prostateBluescript SK- L0438 normalized infant brain total brain brain lafmid BAcDNA L0439 Soares infant brain 1NIB whole Lafmid BA brain L0471 Humanfetal heart, Lambda ZAP Lambda ZAP Express Express L0475 KG1-a LambdaZap KG1-a Lambda Zap Express cDNA library Express (Stratagene) L0483Human pancreatic islet Lambda ZAPII L0518 NCI_CGAP_Pr2 pAMP10 L0519NCI_CGAP_Pr3 pAMP10 L0520 NCI_CGAP_Alv1 alveolar pAMP10 rhabdomyosarcomaL0521 NCI_CGAP_Ew1 Ewing's sarcoma pAMP10 L0529 NCI_CGAP_Pr6 prostatepAMP10 L0530 NCI_CGAP_Pr8 prostate pAMP10 L0581 Stratagene liver liverpBluescript (#937224) SK L0588 Stratagene endothelial pBluescript cell937223 SK- L0591 Stratagene HeLa cell s3 pBluescript 937216 SK- L0592Stratagene hNT neuron pBluescript (#937233) SK- L0593 StratagenepBluescript neuroepithelium SK- (#937231) L0596 Stratagene colon colonpBluescript (#937204) SK- L0598 Morton Fetal Cochlea cochlea earpBluescript SK- L0599 Stratagene lung lung pBluescript (#937210) SK-L0601 Stratagene pancreas pancreas pBluescript (#937208) SK- L0604Stratagene muscle muscle skeletal pBluescript 937209 muscle SK- L0608Stratagene lung lung carcinoma lung NCI-H69 pBluescript carcinoma 937218SK- L0615 22 week old human fetal pBluescriptII liver cDNA library SK(−)L0622 HM1 pcDNAII (Invitrogen) L0623 HM3 pectoral muscle (after pcDNAIImastectomy) (Invitrogen) L0629 NCI_CGAP_Mel3 metastatic melanoma bowelpCMV- to bowel (skin SPORT4 primary) L0638 NCI_CGAP_Brn35 tumor, 5pooled (see brain pCMV- description) SPORT6 L0640 NCI_CGAP_Br18 fourpooled high- breast pCMV- grade tumors, SPORT6 including two prima L0641NCI_CGAP_Co17 juvenile granulosa colon pCMV- tumor SPORT6 L0643NCI_CGAP_Co19 moderately colon pCMV- differentiated SPORT6adenocarcinoma L0644 NCI_CGAP_Co20 moderately colon pCMV- differentiatedSPORT6 adenocarcinoma L0646 NCI_CGAP_Co14 moderately- colon pCMV-differentiated SPORT6 adenocarcinoma L0648 NCI_CGAP_Eso2 squamous cellesophagus pCMV- carcinoma SPORT6 L0649 NCI_CGAP_GU1 2 pooled high-gradegenitourinary pCMV- transitional cell tract SPORT6 tumors L0652NCI_CGAP_Lu27 four pooled poorly- lung pCMV- differentiated SPORT6adenocarcinomas L0653 NCI_CGAP_Lu28 two pooled squamous lung pCMV- cellcarcinomas SPORT6 L0655 NCI_CGAP_Lym12 lymphoma, follicular lymph pCMV-mixed small and node SPORT6 large cell L0657 NCI_CGAP_Ov23 tumor, 5pooled (see ovary pCMV- description) SPORT6 L0659 NCI_CGAP_Pan1adenocarcinoma pancreas pCMV- SPORT6 L0662 NCI_CGAP_Gas4 poorlydifferentiated stomach pCMV- adenocarcinoma with SPORT6 signet r L0663NCI_CGAP_Ut2 moderately- uterus pCMV- differentiated SPORT6 endometrialadenocarcino L0664 NCI_CGAP_Ut3 poorly-differentiated uterus pCMV-endometrial SPORT6 adenocarcinoma, L0665 NCI_CGAP_Ut4 serous papillaryuterus pCMV- carcinoma, high SPORT6 grade, 2 pooled t L0666 NCI_CGAP_Ut1well-differentiated uterus pCMV- endometrial SPORT6 adenocarcinoma, 7L0667 NCI_CGAP_CML1 myeloid cells, 18 whole pCMV- pooled CML cases,blood SPORT6 BCR/ABL rearra L0682 Stanley Frontal NB pool 2 frontal lobe(see brain pCR2.1- description) TOPO (Invitrogen) L0710 NIH_MGC_7 smallcell carcinoma lung MGC3 pOTB7 L0717 Gessler Wilms tumor pSPORT1 L0731Soares_pregnant_uterus_(—) uterus pT7T3-Pac NbHPU L0738 Human colorectalcancer pT7T3D L0740 Soares melanocyte melanocyte pT7T3D 2NbHM(Pharmacia) with a modified polylinker L0741 Soares adult brain brainpT7T3D N2b4HB55Y (Pharmacia) with a modified polylinker L0742 Soaresadult brain brain pT7T3D N2b5HB55Y (Pharmacia) with a modifiedpolylinker L0743 Soares breast 2NbHBst breast pT7T3D (Pharmacia) with amodified polylinker L0744 Soares breast 3NbHBst breast pT7T3D(Pharmacia) with a modified polylinker L0745 Soares retina N2b4HR retinaeye pT7T3D (Pharmacia) with a modified polylinker L0746 Soares retinaN2b5HR retina eye pT7T3D (Pharmacia) with a modified polylinker L0747Soares_fetal_heart_NbHH19W heart pT7T3D (Pharmacia) with a modifiedpolylinker L0748 Soares fetal liver spleen Liver and pT7T3D 1NFLS Spleen(Pharmacia) with a modified polylinker L0749Soares_fetal_liver_spleen_(—) Liver and pT7T3D 1NFLS_S1 Spleen(Pharmacia) with a modified polylinker L0750 Soares_fetal_lung_NbHL19Wlung pT7T3D (Pharmacia) with a modified polylinker L0751 Soares ovarytumor ovarian tumor ovary pT7T3D NbHOT (Pharmacia) with a modifiedpolylinker L0752 Soares_parathyroid_tumor_(—) parathyroid tumorparathyroid pT7T3D NbHPA gland (Pharmacia) with a modified polylinkerL0753 Soares_pineal_gland_N3HPG pineal pT7T3D gland (Pharmacia) with amodified polylinker L0754 Soares placenta Nb2HP placenta pT7T3D(Pharmacia) with a modified polylinker L0755Soares_placenta_8to9weeks_(—) placenta pT7T3D 2NbHP8to9W (Pharmacia)with a modified polylinker L0756 Soares_multiple_sclerosis_(—) multiplesclerosis pT7T3D 2NbHMSP lesions (Pharmacia) with a modified polylinkerV_TYPE L0757 Soares_senescent_fibroblasts_(—) senescent fibroblastpT7T3D NbHSF (Pharmacia) with a modified polylinker V_TYPE L0758Soares_testis_NHT pT7T3D-Pac (Pharmacia) with a modified polylinkerL0759 Soares_total_fetus_Nb2HF8_(—) pT7T3D-Pac 9w (Pharmacia) with amodified polylinker L0761 NCI_CGAP_CLL1 B-cell, chronic pT7T3D-Paclymphotic leukemia (Pharmacia) with a modified polylinker L0763NCI_CGAP_Br2 breast pT7T3D-Pac (Pharmacia) with a modified polylinkerL0764 NCI_CGAP_Co3 colon pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0766 NCI_CGAP_GCB1 germinal center B pT7T3D-Pac cell(Pharmacia) with a modified polylinker L0768 NCI_CGAP_GC4 pooled germcell pT7T3D-Pac tumors (Pharmacia) with a modified polylinker L0769NCI_CGAP_Brn25 anaplastic brain pT7T3D-Pac oligodendroglioma (Pharmacia)with a modified polylinker L0770 NCI_CGAP_Brn23 glioblastoma brainpT7T3D-Pac (pooled) (Pharmacia) with a modified polylinker L0771NCI_CGAP_Co8 adenocarcinoma colon pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0773 NCI_CGAP_Co9 colon tumor RER + colon pT7T3D-Pac(Pharmacia) with a modified polylinker L0774 NCI_CGAP_Kid3 kidneypT7T3D-Pac (Pharmacia) with a modified polylinker L0775 NCI_CGAP_Kid5 2pooled tumors kidney pT7T3D-Pac (clear cell type) (Pharmacia) with amodified polylinker L0776 NCI_CGAP_Lu5 carcinoid lung pT7T3D-Pac(Pharmacia) with a modified polylinker L0777 Soares_NhHMPu_S1 Pooledhuman mixed pT7T3D-Pac melanocyte, fetal (see (Pharmacia) heart, andpregnant below) with a modified polylinker L0779 Soares_NFL_T_GBC_S1pooled pT7T3D-Pac (Pharmacia) with a modified polylinker L0780Soares_NSF_F8_9W_OT_(—) pooled pT7T3D-Pac PA_P_S1 (Pharmacia) with amodified polylinker L0782 NCI_CGAP_Pr21 normal prostate prostatepT7T3D-Pac (Pharmacia) with a modified polylinker L0783 NCI_CGAP_Pr22normal prostate prostate pT7T3D-Pac (Pharmacia) with a modifiedpolylinker L0784 NCI_CGAP_Lei2 leiomyosarcoma soft tissue pT7T3D-Pac(Pharmacia) with a modified polylinker L0785 Barstead spleen spleenpT7T3D-Pac HPLRB2 (Pharmacia) with a modified polylinker L0788NCI_CGAP_Sub2 pT7T3D-Pac (Pharmacia) with a modified polylinker L0789NCI_CGAP_Sub3 pT7T3D-Pac (Pharmacia) with a modified polylinker L0790NCI_CGAP_Sub4 pT7T3D-Pac (Pharmacia) with a modified polylinker L0791NCI_CGAP_Sub5 pT7T3D-Pac (Pharmacia) with a modified polylinker L0794NCI_CGAP_GC6 pooled germ cell pT7T3D-Pac tumors (Pharmacia) with amodified polylinker L0800 NCI_CGAP_Co16 colon tumor, RER+ colonpT7T3D-Pac (Pharmacia) with a modified polylinker L0803 NCI_CGAP_Kid11kidney pT7T3D-Pac (Pharmacia) with a modified polylinker L0804NCI_CGAP_Kid12 2 pooled tumors kidney pT7T3D-Pac (clear cell type)(Pharmacia) with a modified polylinker L0805 NCI_CGAP_Lu24 carcinoidlung pT7T3D-Pac (Pharmacia) with a modified polylinker L0809NCI_CGAP_Pr28 prostate pT7T3D-Pac (Pharmacia) with a modified polylinkerL2991 BN0264 breast_normal puc 18 L3811 NPC pituitary pBluescript sk (-)L3816 HEMBA1 whole embryo, pME18SFL3 mainly head L3825 NT2RM4 NT2pME18SFL3 L3826 NT2RP1 NT2 pUC19FL3 L5565 NCI_CGAP_Brn66 glioblastomawith brain pCMV- probably TP53 SPORT6 mutation and witho L5566NCI_CGAP_Brn70 anaplastic brain pCMV- oligodendroglioma SPORT6.ccdbL5622 NCI_CGAP_Skn3 skin pCMV- SPORT6 L5623 NCI_CGAP_Skn4 squamous cellskin pCMV- carcinoma SPORT6

TABLE 5 OMIM Reference Description 103581 Albright hereditaryosteodystrophy-2 106300 Ankylosing spondylitis 108800 Atrial septaldefect, secundum type 120290 OSMED syndrome, 215150 120290 Sticklersyndrome, type II, 184840 120810 C4 deficiency 120820 C4 deficiency142857 Pemphigoid, susceptibility to 142858 Beryllium disease, chronic,susceptibility to 146150 Hypomelanosis of Ito 150270 Laryngeal adductorparalysis 166800 Otosclerosis 167250 Paget disease of bone 170261 Barelymphocyte syndrome, type I, due to TAP2 deficiency 176270 Prader-Willisyndrome 177900 Psoriasis susceptibility-1 179450 Ragweed sensitivity192340 Diabetes insipidus, neurohypophyseal, 125700 201910 Adrenalhyperplasia, congenital, due to 21-hydroxylase deficiency 210900 Bloomsyndrome 217000 C2 deficiency 218000 Andermann syndrome 222100 Diabetesmellitus, insulin-dependent-1 227220 [Eye color, brown] 233100 [Renalglucosuria] 234200 Neurodegeneration with brain iron accumulation 235200Hemochromatosis 248611 Maple syrup urine disease, type Ib 256550Sialidosis, type I 256550 Sialidosis, type II 600202 Dyslexia, specific,2 600261 Ehlers-Danlos-like syndrome 601623 Angelman syndrome 601800[Hair color, brown] 601868 Deafness, autosomal dominant 13 601889Lymphoma, diffuse large cell 602117 Prader-Willi syndrome 602280Retinitis pigmentosa-14, 600132 602475 Ossification of posteriorlongitudinal ligament of spine

The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

The polypeptides may be in the form of the secreted protein, includingthe mature form, or may be a part of a larger protein, such as a fusionprotein (see below). It is often advantageous to include an additionalamino acid sequence which contains secretory or leader sequences,pro-sequences, sequences which aid in purification, such as multiplehistidine residues, or an additional sequence for stability duringrecombinant production.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, including the secretedpolypeptide, can be substantially purified using techniques describedherein or otherwise known in the art, such as, for example, by theone-step method described in Smith and Johnson, Gene 67:31-40 (1988).Polypeptides of the invention also can be purified from natural,synthetic or recombinant sources using techniques described herein orotherwise known in the art, such as, for example, antibodies of theinvention raised against the secreted protein.

The present invention provides a polynucleotide comprising, oralternatively consisting of, the nucleic acid sequence of SEQ ID NO:X,and/or a cDNA contained in ATCC deposit Z. The present invention alsoprovides a polypeptide comprising, or alternatively, consisting of, thepolypeptide sequence of SEQ ID NO:Y and/or a polypeptide encoded by thecDNA contained in ATCC deposit Z. Polynucleotides encoding a polypeptidecomprising, or alternatively consisting of the polypeptide sequence ofSEQ ID NO:Y and/or a polypeptide sequence encoded by the cDNA containedin ATCC deposit Z are also encompassed by the invention.

Signal Sequences

The present invention also encompasses mature forms of the polypeptidehaving the polypeptide sequence of SEQ ID NO:Y and/or the polypeptidesequence encoded by the cDNA in a deposited clone. Polynucleotidesencoding the mature forms (such as, for example, the polynucleotidesequence in SEQ ID NO:X and/or the polynucleotide sequence contained inthe cDNA of a deposited clone) are also encompassed by the invention.According to the signal hypothesis, proteins secreted by mammalian cellshave a signal or secretary leader sequence which is cleaved from themature protein once export of the growing protein chain across the roughendoplasmic reticulum has been initiated. Most mammalian cells and eveninsect cells cleave secreted proteins with the same specificity.However, in some cases, cleavage of a secreted protein is not entirelyuniform, which results in two or more mature species of the protein.Further, it has long been known that cleavage specificity of a secretedprotein is ultimately determined by the primary structure of thecomplete protein, that is, it is inherent in the amino acid sequence ofthe polypeptide.

Methods for predicting whether a protein has a signal sequence, as wellas the cleavage point for that sequence, are available. For instance,the method of McGeoch, Virus Res. 3:271-286 (1985), uses the informationfrom a short N-terminal charged region and a subsequent uncharged regionof the complete (uncleaved) protein. The method of von Heinje, NucleicAcids Res. 14:4683-4690 (1986) uses the information from the residuessurrounding the cleavage site, typically residues −13 to +2, where +1indicates the amino terminus of the secreted protein. The accuracy ofpredicting the cleavage points of known mammalian secretory proteins foreach of these methods is in the range of 75-80%. (von Heinje, supra.)However, the two methods do not always produce the same predictedcleavage point(s) for a given protein.

In the present case, the deduced amino acid sequence of the secretedpolypeptide was analyzed by a computer program called SignalP (HenrikNielsen et al., Protein Engineering 10:1-6 (1997)), which predicts thecellular location of a protein based on the amino acid sequence. As partof this computational prediction of localization, the methods of McGeochand von Heinje are incorporated. The analysis of the amino acidsequences of the secreted proteins described herein by this programprovided the results shown in Table 1A.

As one of ordinary skill would appreciate, however, cleavage sitessometimes vary from organism to organism and cannot be predicted withabsolute certainty. Accordingly, the present invention provides secretedpolypeptides having a sequence shown in SEQ ID NO:Y which have anN-terminus beginning within 5 residues (i.e., + or −5 residues) of thepredicted cleavage point. Similarly, it is also recognized that in somecases, cleavage of the signal sequence from a secreted protein is notentirely uniform, resulting in more than one secreted species. Thesepolypeptides, and the polynucleotides encoding such polypeptides, arecontemplated by the present invention.

Moreover, the signal sequence identified by the above analysis may notnecessarily predict the naturally occurring signal sequence. Forexample, the naturally occurring signal sequence may be further upstreamfrom the predicted signal sequence. However, it is likely that thepredicted signal sequence will be capable of directing the secretedprotein to the ER. Nonetheless, the present invention provides themature protein produced by expression of the polynucleotide sequence ofSEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA ofa deposited clone, in a mammalian cell (e.g., COS cells, as describedbelow). These polypeptides, and the polynucleotides encoding suchpolypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants

The present invention is directed to variants of the polynucleotidesequence disclosed in SEQ ID NO:X, the complementary strand thereto,and/or the cDNA sequence contained in a deposited clone.

The present invention also encompasses variants of the polypeptidesequence disclosed in SEQ ID NO:Y and/or encoded by a deposited clone.

“Variant” refers to a polynucleotide or polypeptide differing from thepolynucleotide or polypeptide of the present invention, but retainingessential properties thereof. Generally, variants are overall closelysimilar, and, in many regions, identical to the polynucleotide orpolypeptide of the present invention.

The present invention is also directed to nucleic acid molecules whichcomprise, or alternatively consist of, a nucleotide sequence which is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forexample, the nucleotide coding sequence in SEQ ID NO:X or thecomplementary strand thereto, the nucleotide coding sequence containedin a deposited cDNA clone or the complementary strand thereto, anucleotide sequence encoding the polypeptide of SEQ ID NO:Y, anucleotide sequence encoding the polypeptide encoded by the cDNAcontained in a deposited clone, and/or polynucleotide fragments of anyof these nucleic acid molecules (e.g., those fragments describedherein).

Polynucleotides which hybridize to these nucleic acid molecules understringent hybridization conditions or lower stringency conditions arealso encompassed by the invention, as are polypeptides encoded by thesepolynucleotides.

The present invention is also directed to polypeptides which comprise,or alternatively consist of, an amino acid sequence which is at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, thepolypeptide sequence shown in SEQ ID NO:Y, the polypeptide sequenceencoded by the cDNA contained in a deposited clone, and/or polypeptidefragments of any of these polypeptides (e.g., those fragments describedherein).

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of the nucleicacid is identical to the reference sequence except that the nucleotidesequence may include up to five point mutations per each 100 nucleotidesof the reference nucleotide sequence encoding the polypeptide. In otherwords, to obtain a nucleic acid having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. The query sequence may be an entire sequence shown in Table1A, the ORF (open reading frame), or any fragment specified as describedherein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to a nucleotide sequence of the presence invention can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245(1990)). In a sequence alignment the query andsubject sequences are both DNA sequences. An RNA sequence can becompared by converting U's to T's. The result of said global sequencealignment is in percent identity. Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identiy are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, anamino acid sequences shown in Table 1A (SEQ ID NO:Y) or to the aminoacid sequence encoded by cDNA contained in a deposited clone can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245(1990)). In a sequence alignment the query andsubject sequences are either both nucleotide sequences or both aminoacid sequences. The result of said global sequence alignment is inpercent identity. Preferred parameters used in a FASTDB amino acidalignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, JoiningPenalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap-Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

The variants may contain alterations in the coding regions, non-codingregions, or both. Especially preferred are polynucleotide variantscontaining alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedpolypeptide. Nucleotide variants produced by silent substitutions due tothe degeneracy of the genetic code are preferred. Moreover, variants inwhich 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or addedin any combination are also preferred. Polynucleotide variants can beproduced for a variety of reasons, e.g., to optimize codon expressionfor a particular host (change codons in the human mRNA to thosepreferred by a bacterial host such as E. coli).

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985).) These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants may beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the secreted protein without substantial loss ofbiological function. The authors of Ron et al., J. Biol. Chem. 268:2984-2988 (1993), reported variant KGF proteins having heparin bindingactivity even after deleting 3, 8, or 27 amino-terminal amino acidresidues. Similarly, Interferon gamma exhibited up to ten times higheractivity after deleting 8-10 amino acid residues from the carboxyterminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216(1988).)

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine EL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” (See, Abstract.) In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

Thus, the invention further includes polypeptide variants which showsubstantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity. Forexample, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., Science 247:1306-1310(1990), wherein the authors indicate that there are two main strategiesfor studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved. Moreover, tolerated conservative aminoacid substitutions involve replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gln, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly.

Besides conservative amino acid substitution, variants of the presentinvention include (i) substitutions with one or more of thenon-conserved amino acid residues, where the substituted amino acidresidues may or may not be one encoded by the genetic code, or (ii)substitution with one or more of amino acid residues having asubstituent group, or (iii) fusion of the mature polypeptide withanother compound, such as a compound to increase the stability and/orsolubility of the polypeptide (for example, polyethylene glycol), or(iv) fusion of the polypeptide with additional amino acids, such as, forexample, an IgG Fc fusion region peptide, or leader or secretorysequence, or a sequence facilitating purification or (v) fusion of thepolypeptide with another compound, such as albumin (including, but notlimited to, recombinant albumin (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, herein incorporated by reference in theirentirety)). Such variant polypeptides are deemed to be within the scopeof those skilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions ofcharged amino acids with other charged or neutral amino acids mayproduce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of the present invention having anamino acid sequence which contains at least one amino acid substitution,but not more than 50 amino acid substitutions, even more preferably, notmore than 40 amino acid substitutions, still more preferably, not morethan 30 amino acid substitutions, and still even more preferably, notmore than 20 amino acid substitutions. Of course, in order ofever-increasing preference, it is highly preferable for a peptide orpolypeptide to have an amino acid sequence which comprises the aminoacid sequence of the present invention, which contains at least one, butnot more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.In specific embodiments, the number of additions, substitutions, and/ordeletions in the amino acid sequence of the present invention orfragments thereof (e.g., the mature form and/or other fragmentsdescribed herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150,conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

The present invention is also directed to polynucleotide fragments ofthe polynucleotides of the invention.

In the present invention, a “polynucleotide fragment” refers to a shortpolynucleotide having a nucleic acid sequence which: is a portion ofthat contained in a deposited clone, or encoding the polypeptide encodedby the cDNA in a deposited clone; is a portion of that shown in SEQ IDNO:X or the complementary strand thereto, or is a portion of apolynucleotide sequence encoding the polypeptide of SEQ ID NO:Y. Thenucleotide fragments of the invention are preferably at least about 15nt, and more preferably at least about 20 nt, still more preferably atleast about 30 nt, and even more preferably, at least about 40 nt, atleast about 50 nt, at least about 75 nt, or at least about 150 nt inlength. A fragment “at least 20 nt in length,” for example, is intendedto include 20 or more contiguous bases from the cDNA sequence containedin a deposited clone or the nucleotide sequence shown in SEQ ID NO:X. Inthis context “about” includes the particularly recited value, a valuelarger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini. These nucleotide fragments have uses thatinclude, but are not limited to, as diagnostic probes and primers asdiscussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600,2000 nucleotides) are preferred.

Moreover, representative examples, of polynucleotide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, a sequence from about nucleotide number 1-50, 51-100,101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500,501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950,951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250,1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550,1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850,1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:X, orthe complementary strand thereto, or the cDNA contained in a depositedclone. In this context “about” includes the particularly recited ranges,and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides,at either terminus or at both termini. Preferably, these fragmentsencode a polypeptide which has biological activity. More preferably,these polynucleotides can be used as probes or primers as discussedherein. Polynucleotides which hybridize to these nucleic acid moleculesunder stringent hybridization conditions or lower stringency conditionsare also encompassed by the invention, as are polypeptides encoded bythese polynucleotides.

In the present invention, a “polypeptide fragment” refers to an aminoacid sequence which is a portion of that contained in SEQ ID NO:Y orencoded by the cDNA contained in a deposited clone. Protein(polypeptide) fragments may be “free-standing,” or comprised within alarger polypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentscomprising, or alternatively consisting of, from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 tothe end of the coding region. Moreover, polypeptide fragments can beabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150amino acids in length. In this context “about” includes the particularlyrecited ranges or values, and ranges or values larger or smaller byseveral (5, 4, 3, 2, or 1) amino acids, at either extreme or at bothextremes. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Preferred polypeptide fragments include the secreted protein as well asthe mature form. Further preferred polypeptide fragments include thesecreted protein or the mature form having a continuous series ofdeleted residues from the amino or the carboxy terminus, or both. Forexample, any number of amino acids, ranging from 1-60, can be deletedfrom the amino terminus of either the secreted polypeptide or the matureform. Similarly, any number of amino acids, ranging from 1-30, can bedeleted from the carboxy terminus of the secreted protein or matureform. Furthermore, any combination of the above amino and carboxyterminus deletions are preferred. Similarly, polynucleotides encodingthese polypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:Y falling withinconserved domains are specifically contemplated by the presentinvention. Moreover, polynucleotides encoding these domains are alsocontemplated.

Other preferred polypeptide fragments are biologically active fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the polypeptide of thepresent invention. The biological activity of the fragments may includean improved desired activity, or a decreased undesirable activity.Polynucleotides encoding these polypeptide fragments are alsoencompassed by the invention.

Preferably, the polynucleotide fragments of the invention encode apolypeptide which demonstrates a functional activity. By a polypeptidedemonstrating a “functional activity” is meant, a polypeptide capable ofdisplaying one or more known functional activities associated with afull-length (complete) polypeptide of invention protein. Such functionalactivities include, but are not limited to, biological activity,antigenicity [ability to bind (or compete with a polypeptide of theinvention for binding) to an antibody to the polypeptide of theinvention], immunogenicity (ability to generate antibody which binds toa polypeptide of the invention), ability to form multimers withpolypeptides of the invention, and ability to bind to a receptor orligand for a polypeptide of the invention.

The functional activity of polypeptides of the invention, and fragments,variants derivatives, and analogs thereof, can be assayed by variousmethods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length polypeptide of the invention forbinding to an antibody of the polypeptide of the invention, variousimmunoassays known in the art can be used, including but not limited to,competitive and non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

In another embodiment, where a ligand for a polypeptide of the inventionidentified, or the ability of a polypeptide fragment, variant orderivative of the invention to multimerize is being evaluated, bindingcan be assayed, e.g., by means well-known in the art, such as, forexample, reducing and non-reducing gel chromatography, protein affinitychromatography, and affinity blotting. See generally, Phizicky, E., etal., 1995, Microbiol. Rev. 59:94-123. In another embodiment,physiological correlates of binding of a polypeptide of the invention toits substrates (signal transduction) can be assayed.

In addition, assays described herein (see Examples) and otherwise knownin the art may routinely be applied to measure the ability ofpolypeptides of the invention and fragments, variants derivatives andanalogs thereof to elicit related biological activity related to that ofthe polypeptide of the invention (either in vitro or in vivo). Othermethods will be known to the skilled artisan and are within the scope ofthe invention.

Epitopes and Antibodies

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:Y, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in ATCC depositNo. Z or encoded by a polynucleotide that hybridizes to the complementof the sequence of SEQ ID NO:X or contained in ATCC deposit No. Z understringent hybridization conditions or lower stringency hybridizationconditions as defined supra. The present invention further encompassespolynucleotide sequences encoding an epitope of a polypeptide sequenceof the invention (such as, for example, the sequence disclosed in SEQ IDNO:X), polynucleotide sequences of the complementary strand of apolynucleotide sequence encoding an epitope of the invention, andpolynucleotide sequences which hybridize to the complementary strandunder stringent hybridization conditions or lower stringencyhybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof. Antigenic epitopes are useful, for example, to raiseantibodies, including monoclonal antibodies, that specifically bind theepitope. Preferred antigenic epitopes include the antigenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes can be used asthe target molecules in immunoassays. (See, for instance, Wilson et al.,Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg of peptide or carrier protein and Freund's adjuvant or anyother adjuvant known for stimulating an immune response. Several boosterinjections may be needed, for instance, at intervals of about two weeks,to provide a useful titer of anti-peptide antibody which can bedetected, for example, by ELISA assay using free peptide adsorbed to asolid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention (e.g., those comprising animmunogenic or antigenic epitope) can be fused to heterologouspolypeptide sequences. For example, polypeptides of the presentinvention (including fragments or variants thereof), may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof,resulting in chimeric polypeptides. By way of another non-limitingexample, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) may be fused with albumin(including but not limited to recombinant human serum albumin orfragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, herein incorporated by reference in theirentirety)). In a preferred embodiment, polypeptides and/or antibodies ofthe present invention (including fragments or variants thereof) arefused with the mature form of human serum albumin (i.e., amino acids1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0322 094) which is herein incorporated by reference in its entirety. Inanother preferred embodiment, polypeptides and/or antibodies of thepresent invention (including fragments or variants thereof) are fusedwith polypeptide fragments comprising, or alternatively consisting of,amino acid residues 1−x of human serum albumin, where x is an integerfrom 1 to 585 and the albumin fragment has human serum albumin activity.In another preferred embodiment, polypeptides and/or antibodies of thepresent invention (including fragments or variants thereof) are fusedwith polypeptide fragments comprising, or alternatively consisting of,amino acid residues 1−z of human serum albumin, where z is an integerfrom 369 to 419, as described in U.S. Pat. No. 5,766,883 hereinincorporated by reference in its entirety. Polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) may be fused to either the N- or C-terminal end of theheterologous protein (e.g., immunoglobulin Fc polypeptide or human serumalbumin polypeptide). Polynucleotides encoding fusion proteins of theinvention are also encompassed by the invention.

Such fusion proteins as those described above may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). IgG Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion desulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:X and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of SEQ ID NO:Y, and/or an epitope, ofthe present invention (as determined by immunoassays well known in theart for assaying specific antibody-antigen binding). Antibodies of theinvention include, but are not limited to, polyclonal, monoclonal,multispecific, human, humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies (including,e.g., anti-Id antibodies to antibodies of the invention), andepitope-binding fragments of any of the above. The term “antibody,” asused herein, refers to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site that immunospecifically binds anantigen. The immunoglobulin molecules of the invention can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Inpreferred embodiments, the immunoglobulin molecules of the invention areIgG1. In other preferred embodiments, the immunoglobulin molecules ofthe invention are IgG4.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (scFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologs of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M,5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10 ⁻¹² M, ¹⁰⁻¹² M, 5×10⁻¹³ M,10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptoriligandinteractions with the polypeptides of the invention either partially orfully. Preferrably, antibodies of the present invention bind anantigenic epitope disclosed herein, or a portion thereof. The inventionfeatures both receptor-specific antibodies and ligand-specificantibodies. The invention also features receptor-specific antibodieswhich do not prevent ligand binding but prevent receptor activation.Receptor activation (i.e., signaling) may be determined by techniquesdescribed herein or otherwise known in the art. For example, receptoractivation can be determined by detecting the phosphorylation (e.g.,tyrosine or serine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e, by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples (e.g., Example 16). In anon-limiting example, mice can be immunized with a polypeptide of theinvention or a cell expressing such peptide. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theATCC. Hybridomas are selected and cloned by limited dilution. Thehybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding a polypeptide of theinvention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entirety. Humanized antibodies are antibodymolecules from non-human species antibody that binds the desired antigenhaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework regions from a human immunoglobulinmolecule. Often, framework residues in the human framework regions willbe substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:Y.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties ), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NO:Y may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. Further, the polypeptides corresponding to SEQ ID NO:Y maybe fused or conjugated to the above antibody portions to facilitatepurification. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature331:84-86 (1988). The polypeptides of the present invention fused orconjugated to an antibody having disulfide-linked dimeric structures(due to the IgG) may also be more efficient in binding and neutralizingother molecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In manycases, the Fc part in a fusion protein is beneficial in therapy anddiagnosis, and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fc part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al., J. MolecularRecognition 8:52-58 (1995); Johanson et al., J. Biol. Chem.270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be useful as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types. Monoclonal antibodies directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays For Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., 3H or 125I)in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10 ⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO 93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y.(1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99 mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusionproteins. For example, the polypeptide of the present invention, whenfused to a second protein, can be used as an antigenic tag. Antibodiesraised against the polypeptide of the present invention can be used toindirectly detect the second protein by binding to the polypeptide.Moreover, because secreted proteins target cellular locations based ontrafficking signals, the polypeptides of the present invention can beused as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the presentinvention include not only heterologous signal sequences, but also otherheterologous functional regions. The fusion does not necessarily need tobe direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Also, peptide moieties may be added tothe polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routinetechniques in the art.

Moreover, polypeptides of the present invention, including fragments,and specifically epitopes, can be combined with parts of the constantdomain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1,CH2, CH3, and any combination thereof, including both entire domains andportions thereof), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. One reported example describes chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86(1988).) Fusion proteins having disulfide-linked dimeric structures (dueto the IgG) can also be more efficient in binding and neutralizing othermolecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)Polynucleotides comprising or alternatively consisting of nucleic acidswhich encode these fusion proteins are also encompassed by theinvention.

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused tomarker sequences, such as a peptide which facilitates purification ofthe fused polypeptide. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl, Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Another peptide tag useful for purification, the “HA”tag, corresponds to an epitope derived from the influenza hemagglutininprotein. (Wilson et al., Cell 37:767 (1984).)

Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The polynucleotide insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp,phoA and tac promoters, the SV40 early and late promoters and promotersof retroviral LTRs, to name a few. Other suitable promoters will beknown to the skilled artisan. The expression constructs will furthercontain sites for transcription initiation, termination, and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the transcripts expressed by the constructs will preferablyinclude a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPAO815 (all available from Invitrogen, Carlbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking arecombinant vector.

A polypeptide of this invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention, and preferably the secreted form,can also be recovered from: products purified from natural sources,including bodily fluids, tissues and cells, whether directly isolated orcultured; products of chemical synthetic procedures; and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect,and mammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes. Thus, it is wellknown in the art that the N-terminal methionine encoded by thetranslation initiation codon generally is removed with high efficiencyfrom any protein after translation in all eukaryotic cells. While theN-terminal methionine on most proteins also is efficiently removed inmost prokaryotes, for some proteins, this prokaryotic removal process isinefficient, depending on the nature of the amino acid to which theN-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to express thepolypeptide of the present invention in a eukaryotic system. Pichiapastoris is a methylotrophic yeast which can metabolize methanol as itssole carbon source. A main step in the methanol metabolization pathwayis the oxidation of methanol to formaldehyde using O₂. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for O₂. Consequently, in a growth medium depending on methanolas a main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOX1 gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. See,Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, etal., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.15:3859-76 (1987). Thus, a heterologous coding sequence, such as, forexample, a polynucleotide of the present invention, under thetranscriptional regulation of all or part of the AOX1 regulatorysequence is expressed at exceptionally high levels in Pichia yeast grownin the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNAencoding a polypeptide of the invention, as set forth herein, in aPichea yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a protein of the invention by virtue of thestrong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase(PHO) secretory signal peptide (i.e., leader) located upstream of amultiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as,pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG as required.

In another embodiment, high-level expression of a heterologous codingsequence, such as, for example, a polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with the polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences via homologous recombination,resulting in the formation of a new transcription unit (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761,issued Mar. 31, 1998; International Publication No. WO 96/29411,published Sep. 26, 1996; International Publication No. WO 94/12650,published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), thedisclosures of each of which are incorporated by reference in theirentireties).

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W. H. Freeman & Co., N.Y., andHunkapiller et al., Nature, 310:105-111 (1984)). For example, apolypeptide corresponding to a fragment of a polypeptide sequence of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thepolypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modifiedduring or after translation, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. Any of numerous chemical modifications may becarried out by known techniques, including but not limited, to specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.:4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to a proteins via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the protein or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may beaccomplished by any number of means. For example, polyethylene glycolmay be attached to the protein either directly or by an interveninglinker. Linkerless systems for attaching polyethylene glycol to proteinsare described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998);U.S. Pat. Nos. 4,002,531; 5,349,052; WO 95/06058; and WO 98/32466, thedisclosures of each of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monmethoxy polyethyleneglycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction ofprotein with tresylated MPEG, polyethylene glycol is directly attachedto amine groups of the protein. Thus, the invention includesprotein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin WO 98/32466, the entire disclosure of which is incorporated herein byreference. Pegylated protein products produced using the reactionchemistries set out herein are included within the scope of theinvention.

The number of polyethylene glycol moieties attached to each protein ofthe invention (i.e., the degree of substitution) may also vary. Forexample, the pegylated proteins of the invention may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

The polypeptides of the invention may be in monomers or multimers (i.e.,dimers, trimers, tetramers and higher multimers). Accordingly, thepresent invention relates to monomers and multimers of the polypeptidesof the invention, their preparation, and compositions (preferably,Therapeutics) containing them. In specific embodiments, the polypeptidesof the invention are monomers, dimers, trimers or tetramers. Inadditional embodiments, the multimers of the invention are at leastdimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlypolypeptides corresponding to the amino acid sequence of SEQ I) NO:Y orencoded by the cDNA contained in a deposited clone (including fragments,variants, splice variants, and fusion proteins, corresponding to thesepolypeptides as described herein). These homomers may containpolypeptides having identical or different amino acid sequences. In aspecific embodiment, a homomer of the invention is a multimer containingonly polypeptides having an identical amino acid sequence. In anotherspecific embodiment, a homomer of the invention is a multimer containingpolypeptides having different amino acid sequences. In specificembodiments, the multimer of the invention is a homodimer (e.g.,containing polypeptides having identical or different amino acidsequences) or a homotrimer (e.g., containing polypeptides havingidentical and/or different amino acid sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides of differentproteins) in addition to the polypeptides of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence ( e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

In one example, covalent associations are between the heterologoussequence contained in a fusion protein of the invention (see, e.g., U.S.Pat. No. 5,478,925). In a specific example, the covalent associationsare between the heterologous sequence contained in an Fc fusion proteinof the invention (as described herein). In another specific example,covalent associations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, such as for example,oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, (1988)), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimenize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins of the invention are those described in PCT application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a polypeptide of the invention fused to a polypeptidesequence that dimerizes or trimerizes in solution are expressed insuitable host cells, and the resulting soluble multimeric fusion proteinis recovered from the culture supernatant using techniques known in theart.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in Flag® fusion proteins of the invention and anti-Flag®antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hyrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerousways as reagents. The following description should be consideredexemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosomeidentification. There exists an ongoing need to identify new chromosomemarkers, since few chromosome marking reagents, based on actual sequencedata (repeat polymorphisms), are presently available. Eachpolynucleotide of the present invention can be used as a chromosomemarker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primerscan be selected using computer analysis so that primers do not span morethan one predicted exon in the genomic DNA. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the SEQ ID NO:X will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping thepolynucleotides to particular chromosomes. Three or more clones can beassigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeledflow-sorted chromosomes, preselection by hybridization to constructchromosome specific-cDNA libraries and computer mapping techniques (See,e.g., Shuler, Trends Biotechnol 16:456-459 (1998) which is herebyincorporated by reference in its entirety).

Precise chromosomal location of the polynucleotides can also be achievedusing fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verna et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (tomark a single chromosome or a single site on that chromosome) or inpanels (for marking multiple sites and/or multiple chromosomes).

The polynucleotides of the present invention would likewise be usefulfor radiation hybrid mapping, HAPPY mapping, and long range restrictionmapping. For a review of these techniques and others known in the art,see, e.g., Dear, “Genome Mapping: A Practical Approach,” IRL Press atOxford University Press, London (1997); Aydin, J. Mol. Med. 77:691-694(1999); Hacia et al., Mol. Psychiatry 3:483-492 (1998); Herrick et al.,Chromosome Res. 7:409-423 (1999); Hamilton et al., Methods Cell Biol.62:265-280 (2000); and/or Ott, J. Hered. 90:68-70 (1999) each of whichis hereby incorporated by reference in its entirety.

Once a polynucleotide has been mapped to a precise chromosomal location,the physical position of the polynucleotide can be used in linkageanalysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. (Diseasemapping data are found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library).) Assuming 1 megabase mapping resolution and onegene per 20 kb, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50-500 potential causativegenes.

Thus, once coinheritance is established, differences in thepolynucleotide and the corresponding gene between affected andunaffected individuals can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected individuals, but not in normalindividuals, indicates that the mutation may cause the disease. However,complete sequencing of the polypeptide and the corresponding gene fromseveral normal individuals is required to distinguish the mutation froma polymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affectedindividuals as compared to unaffected individuals can be assessed usingpolynucleotides of the present invention. Any of these alterations(altered expression, chromosomal rearrangement, or mutation) can be usedas a diagnostic or prognostic marker.

Thus, the invention also provides a diagnostic method useful duringdiagnosis of a disorder, involving measuring the expression level ofpolynucleotides of the present invention in cells or body fluid from anindividual and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

In still another embodiment, the invention includes a kit for analyzingsamples for the presence of proliferative and/or cancerouspolynucleotides derived from a test subject. In a general embodiment,the kit includes at least one polynucleotide probe containing anucleotide sequence that will specifically hybridize with apolynucleotide of the present invention and a suitable container. In aspecific embodiment, the kit includes two polynucleotide probes definingan internal region of the polynucleotide of the present invention, whereeach probe has one strand containing a 31′ mer-end internal to theregion. In a further embodiment, the probes may be useful as primers forpolymerase chain reaction amplification.

Where a diagnosis of a disorder, has already been made according toconventional methods, the present invention is useful as a prognosticindicator, whereby patients exhibiting enhanced or depressedpolynucleotide of the present invention expression will experience aworse clinical outcome relative to patients expressing the gene at alevel nearer the standard level.

By “measuring the expression level of polynucleotide of the presentinvention” is intended qualitatively or quantitatively measuring orestimating the level of the polypeptide of the present invention or thelevel of the mRNA encoding the polypeptide in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the disorder or beingdetermined by averaging levels from a population of individuals nothaving a disorder. As will be appreciated in the art, once a standardpolypeptide level or mRNA level is known, it can be used repeatedly as astandard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains the polypeptide of the present invention or mRNA. Asindicated, biological samples include body fluids (such as semen, lymph,sera, plasma, urine, synovial fluid and spinal fluid) which contain thepolypeptide of the present invention, and other tissue sources found toexpress the polypeptide of the present invention. Methods for obtainingtissue biopsies and body fluids from mammals are well known in the art.Where the biological sample is to include mRNA, a tissue biopsy is thepreferred source.

The method(s) provided above may preferrably be applied in a diagnosticmethod and/or kits in which polynucleotides and/or polypeptides areattached to a solid support. In one exemplary method, the support may bea “gene chip” or a “biological chip” as described in U.S. Pat. Nos.5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip withpolynucleotides of the present invention attached may be used toidentify polymorphisms between the polynucleotide sequences, withpolynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) wouldbe beneficial in identifying disease loci for many disorders, includingcancerous diseases and conditions. Such a method is described in U.S.Pat. Nos. 5,858,659 and 5,856,104. The U.S. Patents referenced supra arehereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides areincorporated onto a solid support, or gene chip. For the purposes of thepresent invention, a peptide nucleic acid (PNA) is a polyamide type ofDNA analog and the monomeric units for adenine, guanine, thymine andcytosine are available commercially (Perceptive Biosystems). Certaincomponents of DNA, such as phosphorus, phosphorus oxides, or deoxyribosederivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A.Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,666 (1993), PNAs bind specifically and tightly to complementary DNAstrands and are not degraded by nucleases. In fact, PNA binds morestrongly to DNA than DNA itself does. This is probably because there isno electrostatic repulsion between the two strands, and also thepolyamide backbone is more flexible. Because of this, PNA/DNA duplexesbind under a wider range of stringency conditions than DNA/DNA duplexes,making it easier to perform multiplex hybridization. Smaller probes canbe used than with DNA due to the strong binding. In addition, it is morelikely that single base mismatches can be determined with PNA/DNAhybridization because a single mismatch in a PNA/DNA 15-mer lowers themelting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA15-mer duplex. Also, the absence of charge groups in PNA means thathybridization can be done at low ionic strengths and reduce possibleinterference by salt during the analysis.

The present invention is useful for detecting cancer in mammals. Inparticular the invention is useful during diagnosis of pathological cellproliferative neoplasias which include, but are not limited to: acutemyelogenous leukemias including acute monocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute erythroleukemia, acute megakaryocyticleukemia, and acute undifferentiated leukemia, etc.; and chronicmyelogenous leukemias including chronic myelomonocytic leukemia, chronicgranulocytic leukemia, etc. Preferred mammals include monkeys, apes,cats, dogs, cows, pigs, horses, rabbits and humans. Particularlypreferred are humans.

Pathological cell proliferative diseases, disorders, and/or conditionsare often associated with inappropriate activation of proto-oncogenes.(Gelmann, E. P. et al., “The Etiology of Acute Leukemia: MolecularGenetics and Viral Oncology,” in Neoplastic Diseases of the Blood, Vol1., Wiernik, P. H. et al. eds., 161-182 (1985)). Neoplasias are nowbelieved to result from the qualitative alteration of a normal cellulargene product, or from the quantitative modification of gene expressionby insertion into the chromosome of a viral sequence, by chromosomaltranslocation of a gene to a more actively transcribed region, or bysome other mechanism. (Gelmann et al., supra) It is likely that mutatedor altered expression of specific genes is involved in the pathogenesisof some leukemias, among other tissues and cell types. (Gelmann et al.,supra) Indeed, the human counterparts of the oncogenes involved in someanimal neoplasias have been amplified or translocated in some cases ofhuman leukemia and carcinoma. (Gelmann et al., supra)

For example, c-myc expression is highly amplified in the non-lymphocyticleukemia cell line HL-60. When HL-60 cells are chemically induced tostop proliferation, the level of c-myc is found to be downregulated.(International Publication Number WO 91/15580) However, it has beenshown that exposure of HL-60 cells to a DNA construct that iscomplementary to the 5′ end of c-myc or c-myb blocks translation of thecorresponding mRNAs which downregulates expression of the c-myc or c-mybproteins and causes arrest of cell proliferation and differentiation ofthe treated cells. (International Publication Number WO 91/15580;Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al.,Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisanwould appreciate the present invention's usefulness would not be limitedto treatment of proliferative diseases, disorders, and/or conditions ofhematopoietic cells and tissues, in light of the numerous cells and celltypes of varying origins which are known to exhibit proliferativephenotypes.

In addition to the foregoing, a polynucleotide can be used to controlgene expression through triple helix formation or antisense DNA or RNA.Antisense techniques are discussed, for example, in Okano, J. Neurochem.56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRCPress, Boca Raton, Fla. (1988). Triple helix formation isdiscussed in, for instance Lee et al., Nucleic Acids Research 6: 3073(1979); Cooney et al., Science 241: 456 (1988); and Dervan et al.,Science 251: 1360 (1991). Both methods rely on binding of thepolynucleotide to a complementary DNA or RNA. For these techniques,preferred polynucleotides are usually oligonucleotides 20 to 40 bases inlength and complementary to either the region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991) ) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat or prevent disease.

Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell.

The polynucleotides are also useful for identifying individuals fromminute biological samples. The United States military, for example, isconsidering the use of restriction fragment length polymorphism (RFLP)for identification of its personnel. In this technique, an individual'sgenomic DNA is digested with one or more restriction enzymes, and probedon a Southern blot to yield unique bands for identifying personnel. Thismethod does not suffer from the current limitations of “Dog Tags” whichcan be lost, switched, or stolen, making positive identificationdifficult. The polynucleotides of the present invention can be used asadditional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as analternative to RFLP, by determining the actual base-by-base DNA sequenceof selected portions of an individual's genome. These sequences can beused to prepare PCR primers for amplifying and isolating such selectedDNA, which can then be sequenced. Using this technique, individuals canbe identified because each individual will have a unique set of DNAsequences. Once an unique ID database is established for an individual,positive identification of that individual, living or dead, can be madefrom extremely small tissue samples.

Forensic biology also benefits from using DNA-based identificationtechniques as disclosed herein. DNA sequences taken from very smallbiological samples such as tissues, e.g., hair or skin, or body fluids,e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk,lymph, pulmonary sputum or surfactant, urine, fecal matter, etc., can beamplified using PCR. In one prior art technique, gene sequencesamplified from polymorphic loci, such as DQa class II HLA gene, are usedin forensic biology to identify individuals. (Erlich, H., PCRTechnology, Freeman and Co. (1992).) Once these specific polymorphicloci are amplified, they are digested with one or more restrictionenzymes, yielding an identifying set of bands on a Southern blot probedwith DNA corresponding to the DQa class II HLA gene. Similarly,polynucleotides of the present invention can be used as polymorphicmarkers for forensic purposes.

There is also a need for reagents capable of identifying the source of aparticular tissue. Such need arises, for example, in forensics whenpresented with tissue of unknown origin. Appropriate reagents cancomprise, for example, DNA probes or primers specific to particulartissue prepared from the sequences of the present invention. Panels ofsuch reagents can identify tissue by species and/or by organ type. In asimilar fashion, these reagents can be used to screen tissue culturesfor contamination.

In the very least, the polynucleotides of the present invention can beused as molecular weight markers on Southern gels, as diagnostic probesfor the presence of a specific mRNA in a particular cell type, as aprobe to “subtract-out” known sequences in the process of discoveringnovel polynucleotides, for selecting and making oligomers for attachmentto a “gene chip” or other support, to raise anti-DNA antibodies usingDNA immunization techniques, and as an antigen to elicit an immuneresponse.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways.The following description should be considered exemplary and utilizesknown techniques.

A polypeptide of the present invention can be used to assay proteinlevels in a biological sample using antibody-based techniques. Forexample, protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99 mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying secreted protein levels in a biological sample,proteins can also be detected in vivo by imaging. Antibody labels ormarkers for in vivo imaging of protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, 131I, 112In, 99 mTc), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously, or intraperitoneally) into themammal. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99 mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specific protein.In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, whichinvolves (a) assaying the expression of a polypeptide of the presentinvention in cells or body fluid of an individual; (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Moreover, polypeptides of the present invention can be used to treat,prevent, and/or diagnose disease. For example, patients can beadministered a polypeptide of the present invention in an effort toreplace absent or decreased levels of the polypeptide (e.g., insulin),to supplement absent or decreased levels of a different polypeptide(e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repairproteins), to inhibit the activity of a polypeptide (e.g., an oncogeneor tumor supressor), to activate the activity of a polypeptide (e.g., bybinding to a receptor), to reduce the activity of a membrane boundreceptor by competing with it for free ligand (e.g., soluble TNFreceptors used in reducing inflammation), or to bring about a desiredresponse (e.g., blood vessel growth inhibition, enhancement of theimmune response to proliferative cells or tissues).

Similarly, antibodies directed to a polypeptide of the present inventioncan also be used to treat, prevent, and/or diagnose disease. Forexample, administration of an antibody directed to a polypeptide of thepresent invention can bind and reduce overproduction of the polypeptide.Similarly, administration of an antibody can activate the polypeptide,such as by binding to a polypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be usedas molecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart. Polypeptides can also be used to raise antibodies, which in turnare used to measure protein expression from a recombinant cell, as a wayof assessing transformation of the host cell. Moreover, the polypeptidesof the present invention can be used to test the following biologicalactivities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods fortreating or preventing disorders, diseases and conditions. The genetherapy methods relate to the introduction of nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofa polypeptide of the present invention. This method requires apolynucleotide which codes for a polypeptide of the invention thatoperatively linked to a promoter and any other genetic elementsnecessary for the expression of the polypeptide by the target tissue.Such gene therapy and delivery techniques are known in the art, see, forexample, WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide of the invention ex vivo, with the engineered cells thenbeing provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, see Belldegrun et al.,J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., CancerResearch, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al.,Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38(1996)), which are herein incorporated by reference. In one embodiment,the cells which are engineered are arterial cells. The arterial cellsmay be reintroduced into the patient through direct injection to theartery, the tissues surrounding the artery, or through catheterinjection.

As discussed in more detail below, the polynucleotide constructs can bedelivered by any method that delivers injectable materials to the cellsof an animal, such as, injection into the interstitial space of tissues(heart, muscle, skin, lung, liver, and the like). The polynucleotideconstructs may be delivered in a pharmaceutically acceptable liquid oraqueous carrier.

In one embodiment, the polynucleotide of the invention is delivered as anaked polynucleotide. The term “naked” polynucleotide, DNA or RNA refersto sequences that are free from any delivery vehicle that acts toassist, promote or facilitate entry into the cell, including viralsequences, viral particles, liposome formulations, lipofectin orprecipitating agents and the like. However, the polynucleotides of theinvention can also be delivered in liposome formulations and lipofectinformulations and the like can be prepared by methods well known to thoseskilled in the art. Such methods are described, for example, in U.S.Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are hereinincorporated by reference.

The polynucleotide vector constructs of the invention used in the genetherapy method are preferably constructs that will not integrate intothe host genome nor will they contain sequences that allow forreplication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of polynucleotide sequence of the invention.Suitable promoters include adenoviral promoters, such as the adenoviralmajor late promoter; or heterologous promoters, such as thecytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV)promoter; inducible promoters, such as the MMT promoter, themetallothionein promoter; heat shock promoters; the albumin promoter;the ApoAI promoter; human globin promoters; viral thymidine kinasepromoters, such as the Herpes Simplex thymidine kinase promoter;retroviral LTRs; the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter for thepolynucleotides of the invention.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The polynucleotide construct of the invention can be delivered to theinterstitial space of tissues within the an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

For the nakednucleic acid sequence injection, an effective dosage amountof DNA or RNA will be in the range of from about 0.05 mg/kg body weightto about 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the polynucleotide constructs of the inventionare complexed in a liposome preparation. Liposomal preparations for usein the instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA,84:7413-7416 (1987), which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077-6081 (1989), whichis herein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem., 265:10189-10192 (1990), which is hereinincorporated by reference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication NO: WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., Felgner etal., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology, 101:512-527 (1983), which-is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilsonet al., Cell, 17:77 (1979)); ether injection (Deamer et al., Biochim.Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res.Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA,76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad.Sci. USA, 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley etal., J. Biol. Chem., 255:10431 (1980); Szoka et al., Proc. Natl. Acad.Sci. USA, 75:145 (1978); Schaefer-Ridder et al., Science, 215:166(1982)), which are herein incorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No.: 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication NO: WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication NO: WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, usinga retroviral particle containing RNA which comprises a sequence encodingpolypeptides of the invention. Retroviruses from which the retroviralplasmid vectors may be derived include, but are not limited to, MoloneyMurine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, humanimmunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammarytumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, 1:5-14(1990), which is incorporated herein by reference in its entirety. Thevector may transduce the packaging cells through any means known in theart. Such means include, but are not limited to, electroporation, theuse of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding polypeptides of the invention.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express polypeptides of the invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with polynucleotides of the invention contained in an adenovirus vector.Adenovirus can be manipulated such that it encodes and expressespolypeptides of the invention, and at the same time is inactivated interms of its ability to replicate in a normal lytic viral life cycle.Adenovirus expression is achieved without integration of the viral DNAinto the host cell chromosome, thereby alleviating concerns aboutinsertional mutagenesis. Furthermore, adenoviruses have been used aslive enteric vaccines for many years with an excellent safety profile(Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally,adenovirus mediated gene transfer has been demonstrated in a number ofinstances including transfer of alpha-1-antitrypsin and CFRR to thelungs of cotton rats (Rosenfeld et al.,Science, 252:431-434 (1991);Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA,76:6606 (1979)).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel., 3:499-503 (1993); Rosenfeld et al., Cell, 68:143-155 (1992);Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al.,Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692(1993); and U.S. Pat. No.: 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol.,158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructcontaining polynucleotides of the invention is inserted into the AAVvector using standard cloning methods, such as those found in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press(1989). The recombinant AAV vector is then transfected into packagingcells which are infected with a helper virus, using any standardtechnique, including lipofection, electroporation, calcium phosphateprecipitation, etc. Appropriate helper viruses include adenoviruses,cytomegaloviruses, vaccinia viruses, or herpes viruses. Once thepackaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct of the invention. These viral particles are then used totransduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the polynucleotide construct integrated into itsgenome, and will express the desired gene product.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding the polypeptide sequence of interest) via homologousrecombination (see, e.g., U.S. Pat. No.: 5,641,670, issued Jun. 24,1997; International Publication NO: WO 96/29411, published Sep. 26,1996; International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot normally expressed in the cells, or is expressed at a lower levelthan desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the desired endogenous polynucleotide sequence so thepromoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence,

The polynucleotides encoding polypeptides of the present invention maybe administered along with other polynucleotides encoding otherangiongenic proteins. Angiogenic proteins include, but are not limitedto, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C),VEGF-3 (VEGF-B), epidermal growth factor alpha and beta,platelet-derived endothelial cell growth factor, platelet-derived growthfactor, tumor necrosis factor alpha, hepatocyte growth factor, insulinlike growth factor, colony stimulating factor, macrophage colonystimulating factor, granulocyte/macrophage colony stimulating factor,and nitric oxide synthase.

Preferably, the polynucleotide encoding a polypeptide of the inventioncontains a secretory signal sequence that facilitates secretion of theprotein. Typically, the signal sequence is positioned in the codingregion of the polynucleotide to be expressed towards or at the 5′ end ofthe coding region. The signal sequence may be homologous or heterologousto the polynucleotide of interest and may be homologous or heterologousto the cells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers. (Kaneda et al.,Science, 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA,189:11277-11281 (1992), which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian. Therapeuticcompositions of the present invention can be administered to any animal,preferably to mammals and birds. Preferred mammals include humans, dogs,cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humansbeing particularly

Biological Activities

The polynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used in assays to test for one or morebiological activities. If these polynucleotides and polypeptides doexhibit activity in a particular assay, it is likely that thesemolecules may be involved in the diseases associated with the biologicalactivity. Thus, the polynucleotides or polypeptides, or agonists orantagonists could be used to treat the associated disease.

Polynucleotides, translation products and antibodies corresponding tothis gene may be useful for the diagnosis, prognosis, prevention, and/ortreatment of diseases and/or disorders associated with the followingsystems.

Immune Activity

Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, diagnosing and/or prognosing diseases, disorders, and/orconditions of the immune system, by, for example, activating orinhibiting the proliferation, differentiation, or mobilization(chemotaxis) of immune cells. Immune cells develop through a processcalled hematopoiesis, producing myeloid (platelets, red blood cells,neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cellsfrom pluripotent stem cells. The etiology of these immune diseases,disorders, and/or conditions may be genetic, somatic, such as cancer andsome autoimmune diseases, acquired (e.g., by chemotherapy or toxins), orinfectious. Moreover, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention can be used as a markeror detector of a particular immune system disease or disorder.

In another embodiment, a polypeptide of the invention, orpolynucleotides, antibodies, agonists, or antagonists corresponding tothat polypeptide, may be used to treat diseases and disorders of theimmune system and/or to inhibit or enhance an immune response generatedby cells associated with the tissue(s) in which the polypeptide of theinvention is expressed, including one, two, three, four, five, or moretissues disclosed in Table 1A, column 8 (Tissue Distribution LibraryCode).

Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, diagnosing, and/or prognosing immunodeficiencies, includingboth congenital and acquired immunodeficiencies. Examples of B cellimmunodeficiencies in which immunoglobulin levels B cell function and/orB cell numbers are decreased include: X-linked agammaglobulinemia(Bruton's disease), X-linked infantile agammaglobulinemia, X-linkedimmunodeficiency with hyper IgM, non X-linked immunodeficiency withhyper IgM, X-linked lymphoproliferative syndrome (XLP),agammaglobulinemia including congenital and acquired agammaglobulinemia,adult onset agammaglobulinemia, late-onset agammaglobulinemia,dysgammaglobulinemia, hypogammaglobulinemia, unspecifiedhypogammaglobulinemia, recessive agammaglobulinemia (Swiss type),Selective IgM deficiency, selective IgA deficiency, selective IgGsubclass deficiencies, IgG subclass deficiency (with or without IgAdeficiency), Ig deficiency with increased IgM, IgG and IgA deficiencywith increased IgM, antibody deficiency with normal or elevated Igs, Igheavy chain deletions, kappa chain deficiency, B celllymphoproliferative disorder (BLPD), common variable immunodeficiency(CVID), common variable immunodeficiency (CVI) (acquired), and transienthypogammaglobulinemia of infancy.

In specific embodiments, ataxia-telangiectasia or conditions associatedwith ataxia-telangiectasia are treated, prevented, diagnosed, and/orprognosing using the polypeptides or polynucleotides of the invention,and/or agonists or antagonists thereof.

Examples of congenital immunodeficiencies in which T cell and/or B cellfunction and/or number is decreased include, but are not limited to:DiGeorge anomaly, severe combined immunodeficiencies (SCID) (including,but not limited to, X-linked SCID, autosomal recessive SCID, adenosinedeaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency,Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrichsyndrome, and ataxia telangiectasia), thymic hypoplasia, third andfourth pharyngeal pouch syndrome, 22q11.2 deletion, chronicmucocutaneous candidiasis, natural killer cell deficiency (NK),idiopathic CD4+T-lymphocytopenia, immunodeficiency with predominant Tcell defect (unspecified), and unspecified immunodeficiency of cellmediated immunity.

In specific embodiments, DiGeorge anomaly or conditions associated withDiGeorge anomaly are treated, prevented, diagnosed, and/or prognosedusing polypeptides or polynucleotides of the invention, or antagonistsor agonists thereof.

Other immunodeficiencies that may be treated, prevented, diagnosed,and/or prognosed using polypeptides or polynucleotides of the invention,and/or agonists or antagonists thereof, include, but are not limited to,chronic granulomatous disease, Chédiak-Figashi syndrome, myeloperoxidasedeficiency, leukocyte glucose-6-phosphate dehydrogenase deficiency,X-linked lymphoproliferative syndrome (XLP), leukocyte adhesiondeficiency, complement component deficiencies (including C1, C2, C3, C4,C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymicalymphoplasia-aplasia, immunodeficiency with thymoma, severe congenitalleukopenia, dysplasia with immunodeficiency, neonatal neutropenia, shortlimbed dwarfism, and Nezelof syndrome-combined immunodeficiency withIgs.

In a preferred embodiment, the immunodeficiencies and/or conditionsassociated with the immunodeficiencies recited above are treated,prevented, diagnosed and/or prognosed using polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention.

In a preferred embodiment polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention could be used asan agent to boost immunoresponsiveness among immunodeficientindividuals. In specific embodiments, polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present inventioncould be used as an agent to boost immunoresponsiveness among B celland/or T cell immunodeficient individuals.

The polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, diagnosing and/or prognosing autoimmune disorders. Manyautoimmune disorders result from inappropriate recognition of self asforeign material by immune cells. This inappropriate recognition resultsin an immune response leading to the destruction of the host tissue.Therefore, the administration of polynucleotides and polypeptides of theinvention that can inhibit an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune disorders.

Autoimmune diseases or disorders that may be treated, prevented,diagnosed and/or prognosed by polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention include, but arenot limited to, one or more of the following: systemic lupuserythematosus, rheumatoid arthritis, ankylosing spondylitis, multiplesclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmunehemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmunethrombocytopenia purpura, autoimmune neonatal thrombocytopenia,idiopathic thrombocytopenia purpura, purpura (e.g., Henloch-Scoenleinpurpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigusvulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), andinsulin-resistant diabetes mellitus.

Additional disorders that are likely to have an autoimmune componentthat may be treated, prevented, and/or diagnosed with the compositionsof the invention include, but are not limited to, type IIcollagen-induced arthritis, antiphospholipid syndrome, dermatitis,allergic encephalomyelitis, myocarditis, relapsing polychondritis,rheumatic heart disease, neuritis, uveitis ophthalmia,polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, autoimmunepulmonary inflammation, autism, Guillain-Barre Syndrome, insulindependent diabetes mellitus, and autoimmune inflammatory eye disorders.

Additional disorders that are likely to have an autoimmune componentthat may be treated, prevented, diagnosed and/or prognosed with thecompositions of the invention include, but are not limited to,scleroderma with anti-collagen antibodies (often characterized, e.g., bynucleolar and other nuclear antibodies), mixed connective tissue disease(often characterized, e.g., by antibodies to extractable nuclearantigens (e.g., ribonucleoprotein)), polymyositis (often characterized,e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g.,by antiparietal cell, microsomes, and intrinsic factor antibodies),idiopathic Addison's disease (often characterized, e.g., by humoral andcell-mediated adrenal cytotoxicity, infertility (often characterized,e.g., by antispermatozoal antibodies), glomerulonephritis (oftencharacterized, e.g., by glomerular basement membrane antibodies orimmune complexes), bullous pemphigoid (often characterized, e.g., by IgGand complement in basement membrane), Sjogren's syndrome (oftencharacterized, e.g., by multiple tissue antibodies, and/or a specificnonhistone ANA (SS-B)), diabetes mellitus (often characterized, e.g., bycell-mediated and humoral islet cell antibodies), and adrenergic drugresistance (including adrenergic drug resistance with asthma or cysticfibrosis) (often characterized, e.g., by beta-adrenergic receptorantibodies).

Additional disorders that may have an autoimmune component that may betreated, prevented, diagnosed and/or prognosed with the compositions ofthe invention include, but are not limited to, chronic active hepatitis(often characterized, e.g., by smooth muscle antibodies), primarybiliary cirrhosis (often characterized, e.g., by mitochondriaantibodies), other endocrine gland failure (often characterized, e.g.,by specific tissue antibodies in some cases), vitiligo (oftencharacterized, e.g., by melanocyte antibodies), vasculitis (oftencharacterized, e.g., by Ig and complement in vessel walls and/or lowserum complement), post-MI (often characterized, e.g., by myocardialantibodies), cardiotomy syndrome (often characterized, e.g., bymyocardial antibodies), urticaria (often characterized, e.g., by IgG andIgM antibodies to IgE), atopic dermatitis (often characterized, e.g., byIgG and IgM antibodies to IgE), asthma (often characterized, e.g., byIgG and IgM antibodies to IgE), and many other inflammatory,granulomatous, degenerative, and atrophic disorders.

In a preferred embodiment, the autoimmune diseases and disorders and/orconditions associated with the diseases and disorders recited above aretreated, prevented, diagnosed and/or prognosed using for example,antagonists or agonists, polypeptides or polynucleotides, or antibodiesof the present invention. In a specific preferred embodiment, rheumatoidarthritis is treated, prevented, and/or diagnosed using polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention.

In another specific preferred embodiment, systemic lupus erythematosusis treated, prevented, and/or diagnosed using polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention. In another specific preferred embodiment, idiopathicthrombocytopenia purpura is treated, prevented, and/or diagnosed usingpolynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention.

In another specific preferred embodiment IgA nephropathy is treated,prevented, and/or diagnosed using polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present invention.

In a preferred embodiment, the autoimmune diseases and disorders and/orconditions associated with the diseases and disorders recited above aretreated, prevented, diagnosed and/or prognosed using polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention

In preferred embodiments, polypeptides, antibodies, polynucleotidesand/or agonists or antagonists of the present invention are used as aimmunosuppressive agent(s).

Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, prognosing, and/or diagnosing diseases, disorders, and/orconditions of hematopoietic cells. Polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present inventioncould be used to increase differentiation and proliferation ofhematopoietic cells, including the pluripotent stem cells, in an effortto treat or prevent those diseases, disorders, and/or conditionsassociated with a decrease in certain (or many) types hematopoieticcells, including but not limited to, leukopenia, neutropenia, anemia,and thrombocytopenia. Alternatively, Polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present inventioncould be used to increase differentiation and proliferation ofhematopoietic cells, including the pluripotent stem cells, in an effortto treat or prevent those diseases, disorders, and/or conditionsassociated with an increase in certain (or many) types of hematopoieticcells, including but not limited to, histiocytosis.

Allergic reactions and conditions, such as asthma (particularly allergicasthma) or other respiratory problems, may also be treated, prevented,diagnosed and/or prognosed using polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagoniststhereof. Moreover, these molecules can be used to treat, prevent,prognose, and/or diagnose anaphylaxis, hypersensitivity to an antigenicmolecule, or blood group incompatibility.

Additionally, polypeptides or polynucleotides of the invention, and/oragonists or antagonists thereof, may be used to treat, prevent, diagnoseand/or prognose IgE-mediated allergic reactions. Such allergic reactionsinclude, but are not limited to, asthma, rhinitis, and eczema. Inspecific embodiments, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention may be used to modulateIgE concentrations in vitro or in vivo.

Moreover, polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention have uses in the diagnosis,prognosis, prevention, and/or treatment of inflammatory conditions. Forexample, since polypeptides, antibodies, or polynucleotides of theinvention, and/or agonists or antagonists of the invention may inhibitthe activation, proliferation and/or differentiation of cells involvedin an inflammatory response, these molecules can be used to preventand/or treat chronic and acute inflammatory conditions. Suchinflammatory conditions include, but are not limited to, for example,inflammation associated with infection (e.g., septic shock, sepsis, orsystemic inflammatory response syndrome), ischemia-reperfusion injury,endotoxin lethality, complement-mediated hyperacute rejection,nephritis, cytokine or chemokine induced lung injury, inflammatory boweldisease, Crohn's disease, over production of cytokines (e.g., TNF orIL-1.), respiratory disorders (e.g., asthma and allergy);gastrointestinal disorders (e.g., inflammatory bowel disease); cancers(e.g., gastric, ovarian, lung, bladder, liver, and breast); CNSdisorders (e.g., multiple sclerosis; ischemic brain injury and/orstroke, traumatic brain injury, neurodegenerative disorders (e.g.,Parkinson's disease and Alzheimer's disease); AIDS-related dementia; andprion disease); cardiovascular disorders (e.g., atherosclerosis,myocarditis, cardiovascular disease, and cardiopulmonary bypasscomplications); as well as many additional diseases, conditions, anddisorders that are characterized by inflammation (e.g., hepatitis,rheumatoid arthritis, gout, trauma, pancreatitis, sarcoidosis,dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemiclupus erythematosus, diabetes mellitus, and allogenic transplantrejection).

Because inflammation is a fundamental defense mechanism, inflammatorydisorders can effect virtually any tissue of the body. Accordingly,polynucleotides, polypeptides, and antibodies of the invention, as wellas agonists or antagonists thereof, have uses in the treatment oftissue-specific inflammatory disorders, including, but not limited to,adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis,blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis,cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis,dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis,eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis,gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis,laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis,mucitis, myocarditis, myosititis, myringitis, nephritis, neuritis,orchitis, osteochondritis, otitis, pericarditis, peritendonitis,peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis,pulpitis, retinitis, rhinitis, salpingitis, scleritis,sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis,stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis,and vaginitis.

In specific embodiments, polypeptides, antibodies, or polynucleotides ofthe invention, and/or agonists or antagonists thereof, are useful todiagnose, prognose, prevent, and/or treat organ transplant rejectionsand graft-versus-host disease. Organ rejection occurs by host immunecell destruction of the transplanted tissue through an immune response.Similarly, an immune response is also involved in GVHD, but, in thiscase, the foreign transplanted immune cells destroy the host tissues.Polypeptides, antibodies, or polynucleotides of the invention, and/oragonists or antagonists thereof, that inhibit an immune response,particularly the activation, proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventing organrejection or GVHD. In specific embodiments, polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagoniststhereof, that inhibit an immune response, particularly the activation,proliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing experimental allergic and hyperacutexenograft rejection.

In other embodiments, polypeptides, antibodies, or polynucleotides ofthe invention, and/or agonists or antagonists thereof, are useful todiagnose, prognose, prevent, and/or treat immune complex diseases,including, but not limited to, serum sickness, post streptococcalglomerulonephritis, polyarteritis nodosa, and immune complex-inducedvasculitis.

Polypeptides, antibodies, polynucleotides and/or agonists or antagonistsof the invention can be used to treat, detect, and/or prevent infectiousagents. For example, by increasing the immune response, particularlyincreasing the proliferation activation and/or differentiation of Band/or T cells, infectious diseases may be treated, detected, and/orprevented. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention may also directlyinhibit the infectious agent (refer to section of application listinginfectious agents, etc), without necessarily eliciting an immuneresponse.

In another embodiment, polypeptides, antibodies, polynucleotides and/oragonists or antagonists of the present invention are used as a vaccineadjuvant that enhances immune responsiveness to an antigen. In aspecific embodiment, polypeptides, antibodies, polynucleotides and/oragonists or antagonists of the present invention are used as an adjuvantto enhance tumor-specific immune responses.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an adjuvant to enhance anti-viral immune responses.Anti-viral immune responses that may be enhanced using the compositionsof the invention as an adjuvant, include virus and virus associateddiseases or symptoms described herein or otherwise known in the art. Inspecific embodiments, the compositions of the invention are used as anadjuvant to enhance an immune response to a virus, disease, or symptomselected from the group consisting of: AIDS, meningitis, Dengue, EBV,and hepatitis (e.g., hepatitis B). In another specific embodiment, thecompositions of the invention are used as an adjuvant to enhance animmune response to a virus, disease, or symptom selected from the groupconsisting of: HIV/AIDS, respiratory syncytial virus, Dengue, rotavirus,Japanese B encephalitis, influenza A and B, parainfluenza, measles,cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpessimplex, and yellow fever.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an adjuvant to enhance anti-bacterial or anti-fungal immuneresponses. Anti-bacterial or anti-fungal immune responses that may beenhanced using the compositions of the invention as an adjuvant, includebacteria or fungus and bacteria or fungus associated diseases orsymptoms described herein or otherwise known in the art. In specificembodiments, the compositions of the invention are used as an adjuvantto enhance an immune response to a bacteria or fungus, disease, orsymptom selected from the group consisting of: tetanus, Diphtheria,botulism, and meningitis type B.

In another specific embodiment, the compositions of the invention areused as an adjuvant to enhance an immune response to a bacteria orfungus, disease, or symptom selected from the group consisting of:Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonellaparatyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group Bstreptococcus, Shigella spp., Enterotoxigenic Escherichia coli,Enterohemorrhagic E. coli, and Borrelia burgdoferi.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an adjuvant to enhance anti-parasitic immune responses.Anti-parasitic immune responses that may be enhanced using thecompositions of the invention as an adjuvant, include parasite andparasite associated diseases or symptoms described herein or otherwiseknown in the art. In specific embodiments, the compositions of theinvention are used as an adjuvant to enhance an immune response to aparasite. In another specific embodiment, the compositions of theinvention are used as an adjuvant to enhance an immune response toPlasmodium (malaria) or Leishmania.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionmay also be employed to treat infectious diseases including silicosis,sarcoidosis, and idiopathic pulmonary fibrosis; for example, bypreventing the recruitment and activation of mononuclear phagocytes.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an antigen for the generation of antibodies to inhibit orenhance immune mediated responses against polypeptides of the invention.

In one embodiment, polypeptides, antibodies, polynucleotides and/oragonists or antagonists of the present invention are administered to ananimal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig,chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate,and human, most preferably human) to boost the immune system to produceincreased quantities of one or more antibodies (e.g., IgG, IgA, IgM, andIgE), to induce higher affinity antibody production and immunoglobulinclass switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase animmune response.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a stimulator of B cell responsiveness to pathogens.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an activator of T cells.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent that elevates the immune status of an individualprior to their receipt of immunosuppressive therapies.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to induce higher affinity antibodies.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to increase serum immunoglobulin concentrations.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to accelerate recovery of immunocompromisedindividuals.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to boost immunoresponsiveness among agedpopulations and/or neonates.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an immune system enhancer prior to, during, or after bonemarrow transplant and/or other transplants (e.g., allogeneic orxenogeneic organ transplantation). With respect to transplantation,compositions of the invention may be administered prior to, concomitantwith, and/or after transplantation. In a specific embodiment,compositions of the invention are administered after transplantation,prior to the beginning of recovery of T-cell populations. In anotherspecific embodiment, compositions of the invention are firstadministered after transplantation after the beginning of recovery of Tcell populations, but prior to full recovery of B cell populations.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to boost immunoresponsiveness among individualshaving an acquired loss of B cell function. Conditions resulting in anacquired loss of B cell function that may be ameliorated or treated byadministering the polypeptides, antibodies, polynucleotides and/oragonists or antagonists thereof, include, but are not limited to, HIVInfection, AIDS, bone marrow transplant, and B cell chronic lymphocyticleukemia (CLL).

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to boost immunoresponsiveness among individualshaving a temporary immune deficiency. Conditions resulting in atemporary immune deficiency that may be ameliorated or treated byadministering the polypeptides, antibodies, polynucleotides and/oragonists or antagonists thereof, include, but are not limited to,recovery from viral infections (e.g., influenza), conditions associatedwith malnutrition, recovery from infectious mononucleosis, or conditionsassociated with stress, recovery from measles, recovery from bloodtransfusion, and recovery from surgery.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a regulator of antigen presentation by monocytes, dendriticcells, and/or B-cells. In one embodiment, polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present inventionenhance antigen presentation or antagonizes antigen presentation invitro or in vivo. Moreover, in related embodiments, said enhancement orantagonism of antigen presentation may be useful as an anti-tumortreatment or to modulate the immune system.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to direct an individual's immune system towardsdevelopment of a humoral response (i.e. TH2) as opposed to a TH1cellular response.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a means to induce tumor proliferation and thus make it moresusceptible to anti-neoplastic agents. For example, multiple myeloma isa slowly dividing disease and is thus refractory to virtually allanti-neoplastic regimens. If these cells were forced to proliferate morerapidly their susceptibility profile would likely change.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a stimulator of B cell production in pathologies such asAIDS, chronic lymphocyte disorder and/or Common VariableImmunodificiency.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a therapy for generation and/or regeneration of lymphoidtissues following surgery, trauma or genetic defect. In another specificembodiment, polypeptides, antibodies, polynucleotides and/or agonists orantagonists of the present invention are used in the pretreatment ofbone marrow samples prior to transplant.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a gene-based therapy for genetically inherited disordersresulting in immuno-incompetence/immunodeficiency such as observed amongSCID patients.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a means of activating monocytes/macrophages to defendagainst parasitic diseases that effect monocytes such as Leishmania.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a means of regulating secreted cytokines that are elicitedby polypeptides of the invention.

In another embodiment, polypeptides, antibodies, polynucleotides and/oragonists or antagonists of the present invention are used in one or moreof the applications decribed herein, as they may apply to veterinarymedicine.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a means of blocking various aspects of immune responses toforeign agents or self. Examples of diseases or conditions in whichblocking of certain aspects of immune responses may be desired includeautoimmune disorders such as lupus, and arthritis, as well asimmunoresponsiveness to skin allergies, inflammation, bowel disease,injury and diseases/disorders associated with pathogens.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a therapy for preventing the B cell proliferation and Igsecretion associated with autoimmune diseases such as idiopathicthrombocytopenic purpura, systemic lupus erythematosus and multiplesclerosis.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a inhibitor of B and/or T cell migration in endothelialcells. This activity disrupts tissue architecture or cognate responsesand is useful, for example in disrupting immune responses, and blockingsepsis.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a therapy for chronic hypergammaglobulinemia evident in suchdiseases as monoclonal gammopathy of undetermined significance (MGUS),Waldenstrom's disease, related idiopathic monoclonal gammopathies, andplasmacytomas.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionmay be employed for instance to inhibit polypeptide chemotaxis andactivation of macrophages and their precursors, and of neutrophils,basophils, B lymphocytes and some T-cell subsets, e.g., activated andCD8 cytotoxic T cells and natural killer cells, in certain autoimmuneand chronic inflammatory and infective diseases. Examples of autoimmunediseases are described herein and include multiple sclerosis, andinsulin-dependent diabetes.

The polypeptides, antibodies, polynucleotides and/or agonists orantagonists of the present invention may also be employed to treatidiopathic hyper-eosinophilic syndrome by, for example, preventingeosinophil production and migration.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used to enhance or inhibit complement mediated cell lysis.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used to enhance or inhibit antibody dependent cellular cytotoxicity.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionmay also be employed for treating atherosclerosis, for example, bypreventing monocyte infiltration in the artery wall.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionmay be employed to treat adult respiratory distress syndrome (ARDS).

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionmay be useful for stimulating wound and tissue repair, stimulatingangiogenesis, and/or stimulating the repair of vascular or lymphaticdiseases or disorders. Additionally, agonists and antagonists of theinvention may be used to stimulate the regeneration of mucosal surfaces.

In a specific embodiment, polynucleotides or polypeptides, and/oragonists thereof are used to diagnose, prognose, treat, and/or prevent adisorder characterized by primary or acquired immunodeficiency,deficient serum immunoglobulin production, recurrent infections, and/orimmune system dysfunction. Moreover, polynucleotides or polypeptides,and/or agonists thereof may be used to treat or prevent infections ofthe joints, bones, skin, and/or parotid glands, blood-borne infections(e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis),autoimmune diseases (e.g., those disclosed herein), inflammatorydisorders, and malignancies, and/or any disease or disorder or conditionassociated with these infections, diseases, disorders and/ormalignancies) including, but not limited to, CVID, other primary immunedeficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitismedia, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster(e.g., severe herpes zoster), and/or pneumocystis carnii. Other diseasesand disorders that may be prevented, diagnosed, prognosed, and/ortreated with polynucleotides or polypeptides, and/or agonists of thepresent invention include, but are not limited to, HIV infection,HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunctionanemia, thrombocytopenia, and hemoglobinuria.

In another embodiment, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention are used to treat,and/or diagnose an individual having common variable immunodeficiencydisease (“CVID”; also known as “acquired agammaglobulinemia” and“acquired hypogammaglobulinemia”) or a subset of this disease.

In a specific embodiment, polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be used todiagnose, prognose, prevent, and/or treat cancers or neoplasms includingimmune cell or immune tissue-related cancers or neoplasms. Examples ofcancers or neoplasms that may be prevented, diagnosed, or treated bypolynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention include, but are not limited to,acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin'sdisease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL) Chroniclymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt'slymphoma, EBV-transformed diseases, and/or diseases and disordersdescribed in the section entitled “Hyperproliferative Disorders”elsewhere herein.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a therapy for decreasing cellular proliferation of LargeB-cell Lymphomas.

In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a means of decreasing the involvement of B cells and Igassociated with Chronic Myelogenous Leukemia.

In specific embodiments, the compositions of the invention are used asan agent to boost immunoresponsiveness among B cell immunodeficientindividuals, such as, for example, an individual who has undergone apartial or complete splenectomy.

Antagonists of the invention include, for example, binding and/orinhibitory antibodies, antisense nucleic acids, ribozymes or solubleforms of the polypeptides of the present invention (e.g., Fc fusionprotein; see, e.g., Example 9). Agonists of the invention include, forexample, binding or stimulatory antibodies, and soluble forms of thepolypeptides (e.g., Fc fusion proteins; see, e.g., Example 9).polypeptides, antibodies, polynucleotides and/or agonists or antagonistsof the present-invention may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described herein.

In another embodiment, polypeptides, antibodies, polynucleotides and/oragonists or antagonists of the present invention are administered to ananimal (including, but not limited to, those listed above, and alsoincluding transgenic animals) incapable of producing functionalendogenous antibody molecules or having an otherwise compromisedendogenous immune system, but which is capable of producing humanimmunoglobulin molecules by means of a reconstituted or partiallyreconstituted immune system from another animal (see, e.g., publishedPCT Application Nos. WO98/24893, WO96/34096, WO96/33735, andWO91/10741). Administration of polypeptides, antibodies, polynucleotidesand/or agonists or antagonists of the present invention to such animalsis useful for the generation of monoclonal antibodies against thepolypeptides, antibodies, polynucleotides and/or agonists or antagonistsof the present invention in an organ system listed above.

Blood-Related Disorders

The polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be used to modulate hemostatic(the stopping of bleeding) or thrombolytic (clot dissolving) activity.For example, by increasing hemostatic or thrombolytic activity,polynucleotides or polypeptides, and/or agonists or antagonists of thepresent invention could be used to treat or prevent blood coagulationdiseases, disorders, and/or conditions (e.g., afibrinogenemia, factordeficiencies, hemophilia), blood platelet diseases, disorders, and/orconditions (e.g., thrombocytopenia), or wounds resulting from trauma,surgery, or other causes. Alternatively, polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present invention thatcan decrease hemostatic or thrombolytic activity could be used toinhibit or dissolve clotting. These molecules could be important in thetreatment or prevention of heart attacks (infarction), strokes, orscarring.

In specific embodiments, the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be used toprevent, diagnose, prognose, and/or treat thrombosis, arterialthrombosis, venous thrombosis, thromboembolism, pulmonary embolism,atherosclerosis, myocardial infarction, transient ischemic attack,unstable angina. In specific embodiments, the polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention may be used for the prevention of occulsion of saphenousgrafts, for reducing the risk of periprocedural thrombosis as mightaccompany angioplasty procedures, for reducing the risk of stroke inpatients with atrial fibrillation including nonrheumatic atrialfibrillation, for reducing the risk of embolism associated withmechanical heart valves and or mitral valves disease. Other uses for thepolynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention, include, but are not limited to,the prevention of occlusions in extrcorporeal devices (e.g.,intravascular canulas, vascular access shunts in hemodialysis patients,hemodialysis machines, and cardiopulmonary bypass machines).

In another embodiment, a polypeptide of the invention, orpolynucleotides, antibodies, agonists, or antagonists corresponding tothat polypeptide, may be used to prevent, diagnose, prognose, and/ortreat diseases and disorders of the blood and/or blood forming organsassociated with the tissue(s) in which the polypeptide of the inventionis expressed, including one, two, three, four, five, or more tissuesdisclosed in Table 1A, column 8 (Tissue Distribution Library Code).

The polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be used to modulatehematopoietic activity (the formation of blood cells). For example, thepolynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be used to increase thequantity of all or subsets of blood cells, such as, for example,erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g.,basophils, eosinophils, neutrophils, mast cells, macrophages) andplatelets. The ability to decrease the quantity of blood cells orsubsets of blood cells may be useful in the prevention, detection,diagnosis and/or treatment of anemias and leukopenias described below.Alternatively, the polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention may be used to decreasethe quantity of all or subsets of blood cells, such as, for example,erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g.,basophils, eosinophils, neutrophils, mast cells, macrophages) andplatelets. The ability to decrease the quantity of blood cells orsubsets of blood cells may be useful in the prevention, detection,diagnosis and/or treatment of leukocytoses, such as, for exampleeosinophilia.

The polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be used to prevent, treat, ordiagnose blood dyscrasia.

Anemias are conditions in which the number of red blood cells or amountof hemoglobin (the protein that carries oxygen) in them is below normal.Anemia may be caused by excessive bleeding, decreased red blood cellproduction, or increased red blood cell destruction (hemolysis). Thepolynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, and/or diagnosing anemias. Anemias that may be treatedprevented or diagnosed by the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention include irondeficiency anemia, hypochromic anemia, microcytic anemia, chlorosis,hereditary siderob;astic anemia, idiopathic acquired sideroblasticanemia, red cell aplasia, megaloblastic anemia (e.g., pernicious anemia,(vitamin B12 deficiency) and folic acid deficiency anemia), aplasticanemia, hemolytic anemias (e.g., autoimmune helolytic anemia,microangiopathic hemolytic anemia, and paroxysmal nocturnalhemoglobinuria). The polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention may be useful intreating, preventing, and/or diagnosing anemias associated with diseasesincluding but not limited to, anemias associated with systemic lupuserythematosus, cancers, lymphomas, chronic renal disease, and enlargedspleens. The polynucleotides, polypeptides, antibodies, and/or agonistsor antagonists of the present invention may be useful in treating,preventing, and/or diagnosing anemias arising from drug treatments suchas anemias associated with methyldopa, dapsone, and/or sulfadrugs.Additionally, rhe polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention may be useful intreating, preventing, and/or diagnosing anemias associated with abnormalred blood cell architecture including but not limited to, hereditaryspherocytosis, hereditary elliptocytosis, glucose-6-phosphatedehydrogenase deficiency, and sickle cell anemia.

The polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, and/or diagnosing hemoglobin abnormalities, (e.g., thoseassociated with sickle cell anemia, hemoglobin C disease, hemoglobin S-Cdisease, and hemoglobin E disease). Additionally, the polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention may be useful in diagnosing, prognosing, preventing, and/ortreating thalassemias, including, but not limited to major and minorforms of alpha-thalassemia and beta-thalassemia.

In another embodiment, the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be useful indiagnosing, prognosing, preventing, and/or treating bleeding disordersincluding, but not limited to, thrombocytopenia (e.g., idiopathicthrombocytopenic purpura, and thrombotic thrombocytopenic purpura), VonWillebrand's disease, hereditary platelet disorders (e.g., storage pooldisease such as Chediak-Higashi and Hermansky-Pudlak syndromes,thromboxane A2 dysfunction, thromboasthenia, and Bernard-Souliersyndrome), hemolytic-uremic syndrome, hemophelias such as hemophelia Aor Factor VII deficiency and Christmas disease or Factor IX deficiency,Hereditary Hemorhhagic Telangiectsia, also known as Rendu-Osler-Webersyndrome, allergic purpura (Henoch Schonlein purpura) and disseminatedintravascular coagulation.

The effect of the polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention on the clotting time ofblood may be monitored using any of the clotting tests known in the artincluding, but not limited to, whole blood partial thromboplastin time(PTT), the activated partial thromboplastin time (aPTT), the activatedclotting time (ACT), the recalcified activated clotting time, or theLee-White Clotting time.

Several diseases and a variety of drugs can cause platelet dysfunction.Thus, in a specific embodiment, the polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present invention maybe useful in diagnosing, prognosing, preventing, and/or treatingacquired platelet dysfunction such as platelet dysfunction accompanyingkidney failure, leukemia, multiple myeloma, cirrhosis of the liver, andsystemic lupus erythematosus as well as platelet dysfunction associatedwith drug treatments, including treatment with aspirin, ticlopidine,nonsteroidal anti-inflammatory drugs (used for arthritis, pain, andsprains), and penicillin in high doses.

In another embodiment, the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be useful indiagnosing, prognosing, preventing, and/or treating diseases anddisorders characterized by or associated with increased or decreasednumbers of white blood cells. Leukopenia occurs when the number of whiteblood cells decreases below normal. Leukopenias include, but are notlimited to, neutropenia and lymphocytopenia. An increase in the numberof white blood cells compared to normal is known as leukocytosis. Thebody generates increased numbers of white blood cells during infection.Thus, leukocytosis may simply be a normal physiological parameter thatreflects infection. Alternatively, leukocytosis may be an indicator ofinjury or other disease such as cancer. Leokocytoses, include but arenot limited to, eosinophilia, and accumulations of macrophages. Inspecific embodiments, the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be useful indiagnosing, prognosing, preventing, and/or treating leukopenia. In otherspecific embodiments, the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be useful indiagnosing, prognosing, preventing, and/or treating leukocytosis.

Leukopenia may be a generalized decreased in all types of white bloodcells, or may be a specific depletion of particular types of white bloodcells. Thus, in specific embodiments, the polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present invention maybe useful in diagnosing, prognosing, preventing, and/or treatingdecreases in neutrophil numbers, known as neutropenia. Neutropenias thatmay be diagnosed, prognosed, prevented, and/or treated by thepolynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention include, but are not limited to,infantile genetic agranulocytosis, familial neutropenia, cyclicneutropenia, neutropenias resulting from or associated with dietarydeficiencies (e.g., vitamin B 12 deficiency or folic acid deficiency),neutropenias resulting from or associated with drug treatments (e.g.,antibiotic regimens such as penicillin treatment, sulfonamide treatment,anticoagulant treatment, anticonvulsant drugs, anti-thyroid drugs, andcancer chemotherapy), and neutropenias resulting from increasedneutrophil destruction that may occur in association with some bacterialor viral infections, allergic disorders, autoimmune diseases, conditionsin which an individual has an enlarged spleen (e.g., Felty syndrome,malaria and sarcoidosis), and some drug treatment regimens.

The polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in diagnosing,prognosing, preventing, and/or treating lymphocytopenias (decreasednumbers of B and/or T lymphocytes), including, but not limitedlymphocytopenias resulting from or associated with stress, drugtreatments (e.g., drug treatment with corticosteroids, cancerchemotherapies, and/or radiation therapies), AIDS infection and/or otherdiseases such as, for example, cancer, rheumatoid arthritis, systemiclupus erythematosus, chronic infections, some viral infections and/orhereditary disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich Syndome,severe combined immunodeficiency, ataxia telangiectsia).

The polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in diagnosing,prognosing, preventing, and/or treating diseases and disordersassociated with macrophage numbers and/or macrophage function including,but not limited to, Gaucher's disease, Niemann-Pick disease,Letterer-Siwe disease and Hand-Schuller-Christian disease.

In another embodiment, the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be useful indiagnosing, prognosing, preventing, and/or treating diseases anddisorders associated with eosinophil numbers and/or eosinophil functionincluding, but not limited to, idiopathic hypereosinophilic syndrome,eosinophilia-myalgia syndrome, and Hand-Schuller-Christian disease.

In yet another embodiment, the polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present invention maybe useful in diagnosing, prognosing, preventing, and/or treatingleukemias and lymphomas including, but not limited to, acute lymphocytic(lymphpblastic) leukemia (ALL), acute myeloid (myelocytic, myelogenous,myeloblastic, or myelomonocytic) leukemia, chronic lymphocytic leukemia(e.g., B cell leukemias, T cell leukemias, Sezary syndrome, and Hairycell leukenia), chronic myelocytic (myeloid, myelogenous, orgranulocytic) leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma,Burkitt's lymphoma, and mycosis fungoides.

In other embodiments, the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be useful indiagnosing, prognosing, preventing, and/or treating diseases anddisorders of plasma cells including, but not limited to, plasma celldyscrasias, monoclonal gammaopathies, monoclonal gammopathies ofundetermined significance, multiple myeloma, macroglobulinemia,Waldenstrom's macroglobulinemia, cryoglobulinemia, and Raynaud'sphenomenon.

In other embodiments, the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be useful intreating, preventing, and/or diagnosing myeloproliferative disorders,including but not limited to, polycythemia vera, relative polycythemia,secondary polycythemia, myelofibrosis, acute myelofibrosis, agnogenicmyelod metaplasia, thrombocythemia, (including both primary and secondaythrombocythemia) and chronic myelocytic leukemia.

In other embodiments, the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be useful asa treatment prior to surgery, to increase blood cell production.

In other embodiments, the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be useful asan agent to enhance the migration, phagocytosis, superoxide production,antibody dependent cellular cytotoxicity of neutrophils, eosionophilsand macrophages.

In other embodiments, the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be useful asan agent to increase the number of stem cells in circulation prior tostem cells pheresis. In another specific embodiment, thepolynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful as an agent toincrease the number of stem cells in circulation prior to plateletpheresis.

In other embodiments, the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be useful asan agent to increase cytokine production.

In other embodiments, the polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be useful inpreventing, diagnosing, and/or treating primary hematopoietic disorders.

Hyperproliferative Disorders

In certain embodiments, polynucleotides or polypeptides, or agonists orantagonists of the present invention can be used to treat or detecthyperproliferative disorders, including neoplasms. Polynucleotides orpolypeptides, or agonists or antagonists of the present invention mayinhibit the proliferation of the disorder through direct or indirectinteractions. Alternatively, Polynucleotides or polypeptides, oragonists or antagonists of the present invention may proliferate othercells which can inhibit the hyperproliferative disorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

Examples of hyperproliferative disorders that can be treated or detectedby polynucleotides or polypeptides, or agonists or antagonists of thepresent invention include, but are not limited to neoplasms located inthe: colon, abdomen, bone, breast, digestive system, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous (centraland peripheral), lymphatic system, pelvis, skin, soft tissue, spleen,thorax, and urogenital tract.

Similarly, other hyperproliferative disorders can also be treated ordetected by polynucleotides or polypeptides, or agonists or antagonistsof the present invention. Examples of such hyperproliferative disordersinclude, but are not limited to: Acute Childhood Lymphoblastic Leukemia,Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute MyeloidLeukemia, Adrenocortical Carcinoma, Adult (Primary) HepatocellularCancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia,Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin'sLymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma,Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-RelatedLymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile DuctCancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors,Breast Cancer, Cancer of the Renal Pelvis and Ureter, Central NervousSystem (Primary) Lymphoma, Central Nervous System Lymphoma, CerebellarAstrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary)Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood AcuteLymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, ChildhoodBrain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood CerebralAstrocytoma, Childhood Extracranial Germ Cell Tumors, ChildhoodHodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamicand Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, ChildhoodMedulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal andSupratentorial Primitive Neuroectodermal Tumors, Childhood Primary LiverCancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma,Childhood Visual Pathway and Hypothalamic Glioma, Chronic LymphocyticLeukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-CellLymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer,Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma andRelated Tumors, Exocrine Pancreatic Cancer, Extracranial Germ CellTumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, EyeCancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer,Gastric Cancer, Gastrointestinal Carcinoid Tumor, GastrointestinalTumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy CellLeukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin'sDisease, Hodgkin's Lymphoma, Hypergammaglobulinemia, HypopharyngealCancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma,Islet Cell Pancreatic, Cancer, Kaposi's Sarcoma, Kidney Cancer,Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer,Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer,Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma,Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, MetastaticPrimary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, MultipleMyeloma, Multiple Myeloma/Plasma Cell Neoplasm, MyelodysplasticSyndrome, Myelogenous Leukemia, Myeloid Leukemia, MyeloproliferativeDisorders, Nasal Cavity and Paranasal Sinus Cancer, NasopharyngealCancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy,Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult PrimaryMetastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/MalignantFibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian EpithelialCancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor,Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, PenileCancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/MultipleMyeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer,Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis andUreter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell LungCancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

In another preferred embodiment, polynucleotides or polypeptides, oragonists or antagonists of the present invention are used to diagnose,prognose, prevent, and/or treat premalignant conditions and to preventprogression to a neoplastic or malignant state, including but notlimited to those disorders described above. Such uses are indicated inconditions known or suspected of preceding progression to neoplasia orcancer, in particular, where non-neoplastic cell growth consisting ofhyperplasia, metaplasia, or most particularly, dysplasia has occurred(for review of such abnormal growth conditions, see Robbins and Angell,1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp.68-79.)

Hyperplasia is a form of controlled cell proliferation, involving anincrease in cell number in a tissue or organ, without significantalteration in structure or function. Hyperplastic disorders which can bediagnosed, prognosed, prevented, and/or treated with compositions of theinvention (including polynucleotides, polypeptides, agonists orantagonists) include, but are not limited to, angiofollicularmediastinal lymph node hyperplasia, angiolymphoid hyperplasia witheosinophilia, atypical melanocytic hyperplasia, basal cell hyperplasia,benign giant lymph node hyperplasia, cementum hyperplasia, congenitaladrenal hyperplasia, congenital sebaceous hyperplasia, cystichyperplasia, cystic hyperplasia of the breast, denture hyperplasia,ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia,focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibroushyperplasia, inflammatory papillary hyperplasia, intravascular papillaryendothelial hyperplasia, nodular hyperplasia of prostate, nodularregenerative hyperplasia, pseudoepitheliomatous hyperplasia, senilesebaceous hyperplasia, and verrucous hyperplasia.

Metaplasia is a form of controlled cell growth in which one type ofadult or fully differentiated cell substitutes for another type of adultcell. Metaplastic disorders which can be diagnosed, prognosed,prevented, and/or treated with compositions of the invention (includingpolynucleotides, polypeptides, agonists or antagonists) include, but arenot limited to, agnogenic myeloid metaplasia, apocrine metaplasia,atypical metaplasia, autoparenchymatous metaplasia, connective tissuemetaplasia, epithelial metaplasia, intestinal metaplasia, metaplasticanemia, metaplastic ossification, metaplastic polyps, myeloidmetaplasia, primary myeloid metaplasia, secondary myeloid metaplasia,squamous metaplasia, squamous metaplasia of amnion, and symptomaticmyeloid metaplasia.

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where there exists chronicirritation or inflammation. Dysplastic disorders which can be diagnosed,prognosed, prevented, and/or treated with compositions of the invention(including polynucleotides, polypeptides, agonists or antagonists)include, but are not limited to, anhidrotic ectodermal dysplasia,anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigitaldysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervicaldysplasia, chondroectodermal dysplasia, cleidocranial dysplasia,congenital ectodermal dysplasia, craniodiaphysial dysplasia,craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentindysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia,encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia,dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata,epithelial dysplasia, faciodigitogenital dysplasia, familial fibrousdysplasia of jaws, familial white folded dysplasia, fibromusculardysplasia, fibrous dysplasia of bone, florid osseous dysplasia,hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia,hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia, mammarydysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondinidysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia,multiple epiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, ophthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be diagnosed, prognosed,prevented, and/or treated with compositions of the invention (includingpolynucleotides, polypeptides, agonists or antagonists) include, but arenot limited to, benign dysproliferative disorders (e.g., benign tumors,fibrocystic conditions, tissue hypertrophy, intestinal polyps, colonpolyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In another embodiment, a polypeptide of the invention, orpolynucleotides, antibodies, agonists, or antagonists corresponding tothat polypeptide, may be used to diagnose and/or prognose disordersassociated with the tissue(s) in which the polypeptide of the inventionis expressed, including one, two, three, four, five, or more tissuesdisclosed in Table 1A, column 8 (Tissue Distribution Library Code).

In another embodiment, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention conjugated to a toxinor a radioactive isotope, as described herein, may be used to treatcancers and neoplasms, including, but not limited to those describedherein. In a further preferred embodiment, polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention conjugated to a toxin or a radioactive isotope, as describedherein, may be used to treat acute myelogenous leukemia.

Additionally, polynucleotides, polypeptides, and/or agonists orantagonists of the invention may affect apoptosis, and therefore, wouldbe useful in treating a number of diseases associated with increasedcell survival or the inhibition of apoptosis. For example, diseasesassociated with increased cell survival or the inhibition of apoptosisthat could be diagnosed, prognosed, prevented, and/or treated bypolynucleotides, polypeptides, and/or agonists or antagonists of theinvention, include cancers (such as follicular lymphomas, carcinomaswith p53 mutations, and hormone-dependent tumors, including, but notlimited to colon cancer, cardiac tumors, pancreatic cancer, melanoma,retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicularcancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection.

In preferred embodiments, polynucleotides, polypeptides, and/or agonistsor antagonists of the invention are used to inhibit growth, progression,and/or metastasis of cancers, in particular those listed above.

Additional diseases or conditions associated with increased cellsurvival that could be diagnosed, prognosed, prevented, and/or treatedby polynucleotides, polypeptides, and/or agonists or antagonists of theinvention, include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, emangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis that could be diagnosed,prognosed, prevented, and/or treated by polynucleotides, polypeptides,and/or agonists or antagonists of the invention, include AIDS;neurodegenerative disorders (such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellardegeneration and brain tumor or prior associated disease); autoimmunedisorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (e.g., hepatitis related liver injury, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, cachexiaand anorexia.

Hyperproliferative diseases and/or disorders that could be diagnosed,prognosed, prevented, and/or treated by polynucleotides, polypeptides,and/or agonists or antagonists of the invention, include, but are notlimited to, neoplasms located in the liver, abdomen, bone, breast,digestive system, pancreas, peritoneum, endocrine glands (adrenal,parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, headand neck, nervous system (central and peripheral), lymphatic system,pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.

Similarly, other hyperproliferative disorders can also be diagnosed,prognosed, prevented, and/or treated by polynucleotides, polypeptides,and/or agonists or antagonists of the invention. Examples of suchhyperproliferative disorders include, but are not limited to:hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia,Gaucher's Disease, histiocytosis, and any other hyperproliferativedisease, besides neoplasia, located in an organ system listed above.

Another preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating cellproliferative disorders by inserting into an abnormally proliferatingcell a polynucleotide of the present invention, wherein saidpolynucleotide represses said expression.

Another embodiment of the present invention provides a method oftreating cell-proliferative disorders in individuals comprisingadministration of one or more active gene copies of the presentinvention to an abnormally proliferating cell or cells. In a preferredembodiment, polynucleotides of the present invention is a DNA constructcomprising a recombinant expression vector effective in expressing a DNAsequence encoding said polynucleotides. In another preferred embodimentof the present invention, the DNA construct encoding the polynucleotidesof the present invention is inserted into cells to be treated utilizinga retrovirus, or more preferably an adenoviral vector (See G J. Nabel,et. al., PNAS 1999 96: 324-326, which is hereby incorporated byreference). In a most preferred embodiment, the viral vector isdefective and will not transform non-proliferating cells, onlyproliferating cells. Moreover, in a preferred embodiment, thepolynucleotides of the present invention inserted into proliferatingcells either alone, or in combination with or fused to otherpolynucleotides, can then be modulated via an external stimulus (i.e.magnetic, specific small molecule, chemical, or drug administration,etc.), which acts upon the promoter upstream of said polynucleotides toinduce expression of the encoded protein product. As such the beneficialtherapeutic affect of the present invention may be expressly modulated(i.e. to increase, decrease, or inhibit expression of the presentinvention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressingexpression of oncogenic genes or antigens. By “repressing expression ofthe oncogenic genes” is intended the suppression of the transcription ofthe gene, the degradation of the gene transcript (pre-message RNA), theinhibition of splicing, the destruction of the messenger RNA, theprevention of the post-translational modifications of the protein, thedestruction of the protein, or the inhibition of the normal function ofthe protein.

For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directlyto cell proliferative disorder/disease sites in internal organs, bodycavities and the like by use of imaging devices used to guide aninjecting needle directly to the disease site. The polynucleotides ofthe present invention may also be administered to disease sites at thetime of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease ordisorder, affecting any one or any combination of organs, cavities, orbody parts, which is characterized by single or multiple local abnormalproliferations of cells, groups of cells, or tissues, whether benign ormalignant.

Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapieswhich involve administering of anti-polypeptides and anti-polynucleotideantibodies to a mammalian, preferably human, patient for treating one ormore of the described disorders. Methods for producing anti-polypeptidesand anti-polynucleotide antibodies polyclonal and monoclonal antibodiesare described in detail elsewhere herein. Such antibodies may beprovided in pharmaceutically acceptable compositions as known in the artor as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the presentinvention are useful for treating a subject having or developing cellproliferative and/or differentiation disorders as described herein. Suchtreatment comprises administering a single or multiple doses of theantibody, or a fragment, derivative, or a conjugate thereof.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example., which serveto increase the number or activity of effector cells which interact withthe antibodies.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragements thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides,including fragements thereof. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M,10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M,10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M,and 10⁻¹⁵M.

Moreover, polypeptides of the present invention are useful in inhibitingthe angiogenesis of proliferative cells or tissues, either alone, as aprotein fusion, or in combination with other polypeptides directly orindirectly, as described elsewhere herein. In a most preferredembodiment, said anti-angiogenesis effect may be achieved indirectly,for example, through the inhibition of hematopoietic, tumor-specificcells, such as tumor-associated macrophages (See Joseph I B, et al. JNatl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated byreference). Antibodies directed to polypeptides or polynucleotides ofthe present invention may also result in inhibition of angiogenesisdirectly, or indirectly (See Witte L, et al., Cancer Metastasis Rev.17(2):155-61 (1998), which is hereby incorporated by reference)).

Polypeptides, including protein fusions, of the present invention, orfragments thereof may be useful in inhibiting proliferative cells ortissues through the induction of apoptosis. Said polypeptides may acteither directly, or indirectly to induce apoptosis of proliferativecells and tissues, for example in the activation of a death-domainreceptor, such as tumor necrosis factor (TNF) receptor-1, CD95(Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) andTNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (SeeSchulze-Osthoff K, et. al., Eur J Biochem 254(3):439-59 (1998), which ishereby incorporated by reference). Moreover, in another preferredembodiment of the present invention, said polypeptides may induceapoptosis through other mechanisms, such as in the activation of otherproteins which will activate apoptosis, or through stimulating theexpression of said proteins, either alone or in combination with smallmolecule drugs or adjuviants, such as apoptonin, galectins,thioredoxins, anti-inflammatory proteins (See for example, Mutat Res400(1-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998), Chem BiolInteract. April 24; 111-112:23-34 (1998), J Mol Med.76(6):402-12 (1998),Int J Tissue React;20(1):3-15 (1998), which are all hereby incorporatedby reference).

Polypeptides, including protein fusions to, or fragments thereof, of thepresent invention are useful in inhibiting the metastasis ofproliferative cells or tissues. Inhibition may occur as a direct resultof administering polypeptides, or antibodies directed to saidpolypeptides as described elsewhere herein, or indirectly, such asactivating the expression of proteins known to inhibit metastasis, forexample alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol1998;231:125-41, which is hereby incorporated by reference). Suchtherapeutic affects of the present invention may be achieved eitheralone, or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of deliveringcompositions containing the polypeptides of the invention (e.g.,compositions containing polypeptides or polypeptide antibodes associatedwith heterologous polypeptides, heterologous nucleic acids, toxins, orprodrugs) to targeted cells expressing the polypeptide of the presentinvention. Polypeptides or polypeptide antibodes of the invention may beassociated with with heterologous polypeptides, heterologous nucleicacids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/orcovalent interactions.

Polypeptides, protein fusions to, or fragments thereof, of the presentinvention are useful in enhancing the immunogenicity and/or antigenicityof proliferating cells or tissues, either directly, such as would occurif the polypeptides of the present invention ‘vaccinated’ the immuneresponse to respond to proliferative antigens and immunogens, orindirectly, such as in activating the expression of proteins known toenhance the immune response (e.g. chemokines), to said antigens andimmunogens.

Renal Disorders

Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention, may be used to treat, prevent,diagnose, and/or prognose disorders of the renal system. Renal disorderswhich can be diagnosed, prognosed, prevented, and/or treated withcompositions of the invention include, but are not limited to, kidneyfailure, nephritis, blood vessel disorders of kidney, metabolic andcongenital kidney disorders, urinary disorders of the kidney, autoimmunedisorders, sclerosis and necrosis, electrolyte imbalance, and kidneycancers.

Kidney diseases which can be diagnosed, prognosed, prevented, and/ortreated with compositions of the invention include, but are not limitedto, acute kidney failure, chronic kidney failure, atheroembolic renalfailure, end-stage renal disease, inflammatory diseases of the kidney(e.g., acute glomerulonephritis, postinfectious glomerulonephritis,rapidly progressive glomerulonephritis, nephrotic syndrome, membranousglomerulonephritis, familial nephrotic syndrome, membranoproliferativeglomerulonephritis I and II, mesangial proliferative glomerulonephritis,chronic glomerulonephritis, acute tubulointerstitial nephritis, chronictubulointerstitial nephritis, acute post-streptococcalglomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronicnephritis, interstitial nephritis, and post-streptococcalglomerulonephritis), blood vessel disorders of the kidneys (e.g., kidneyinfarction, atheroembolic kidney disease, cortical necrosis, malignantnephrosclerosis, renal vein thrombosis, renal underperfusion, renalretinopathy, renal ischemia-reperfusion, renal artery embolism, andrenal artery stenosis), and kidney disorders resulting form urinarytract disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renallithiasis, nephrolithiasis), reflux nephropathy, urinary tractinfections, urinary retention, and acute or chronic unilateralobstructive uropathy.)

In addition, compositions of the invention can be used to diagnose,prognose, prevent, and/or treat metabolic and congenital disorders ofthe kidney (e.g., uremia, renal amyloidosis, renal osteodystrophy, renaltubular acidosis, renal glycosuria, nephrogenic diabetes insipidus,cystinuria, Fanconi's syndrome, renal fibrocystic osteosis (renalrickets), Hartnup disease, Bartter's syndrome, Liddle's syndrome,polycystic kidney disease, medullary cystic disease, medullary spongekidney, Alport's syndrome, nail-patella syndrome, congenital nephroticsyndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy,nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones,and membranous nephropathy), and autoimmune disorders of the kidney(e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome, IgAnephropathy, and IgM mesangial proliferative glomerulonephritis).

Compositions of the invention can also be used to diagnose, prognose,prevent, and/or treat sclerotic or necrotic disorders of the kidney(e.g., glomerulosclerosis, diabetic nephropathy, focal segmentalglomerulosclerosis (FSGS), necrotizing glomerulonephritis, and renalpapillary necrosis), cancers of the kidney (e.g., nephroma,hypernephroma, nephroblastoma, renal cell cancer, transitional cellcancer, renal adenocarcinoma, squamous cell cancer, and Wilm's tumor),and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema,hydronephritis, proteinuria, hyponatremia, hypernatremia, hypokalemia,hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, andhyperphosphatemia).

Polypeptides may be administered using any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,biolistic injectors, particle accelerators, gelfoam sponge depots, othercommercially available depot materials, osmotic pumps, oral orsuppositorial solid pharmaceutical formulations, decanting or topicalapplications during surgery, aerosol delivery. Such methods are known inthe art. Polypeptides may be administered as part of a Therapeutic,described in more detail below. Methods of delivering polynucleotidesare described in more detail herein.

Cardiovascular Disorders

Polynucleotides or polypeptides, or agonists or antagonists of thepresent invention, may be used to treat, prevent, diagnose, and/orprognose cardiovascular disorders, including, but not limited to,peripheral artery disease, such as limb ischemia.

Cardiovascular disorders include, but are not limited to, cardiovascularabnormalities, such as arterio-arterial fistula, arteriovenous fistula,cerebral arteriovenous malformations, congenital heart defects,pulmonary atresia, and Scimitar Syndrome. Congenital heart defectsinclude, but are not limited to, aortic coarctation, cor triatriatum,coronary vessel anomalies, crisscross heart, dextrocardia, patent ductusarteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic leftheart syndrome, levocardia, tetralogy of fallot, transposition of greatvessels, double outlet right ventricle, tricuspid atresia, persistenttruncus arteriosus, and heart septal defects, such as aortopulmonaryseptal defect, endocardial cushion defects, Lutembacher's Syndrome,trilogy of Fallot, ventricular heart septal defects.

Cardiovascular disorders also include, but are not limited to, heartdisease, such as arrhythmias, carcinoid heart disease, high cardiacoutput, low cardiac output, cardiac tamponade, endocarditis (includingbacterial), heart aneurysm, cardiac arrest, congestive heart failure,congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, hearthypertrophy, congestive cardiomyopathy, left ventricular hypertrophy,right ventricular hypertrophy, post-infarction heart rupture,ventricular septal rupture, heart valve diseases, myocardial diseases,myocardial ischemia, pericardial effusion, pericarditis (includingconstrictive and tuberculous), pneumopericardium, postpericardiotomysyndrome, pulmonary heart disease, rheumatic heart disease, ventriculardysfunction, hyperemia, cardiovascular pregnancy complications, ScimitarSyndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include, but are not limited to, sinus arrhythmia, atrialfibrillation, atrial flutter, bradycardia, extrasystole, Adams-StokesSyndrome, bundle-branch block, sinoatrial block, long QT syndrome,parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitationsyndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome,tachycardias, and ventricular fibrillation. Tachycardias includeparoxysmal tachycardia, supraventricular tachycardia, acceleratedidioventricular rhythm, atrioventricular nodal reentry tachycardia,ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrialnodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, andventricular tachycardia.

Heart valve diseases include, but are not limited to, aortic valveinsufficiency, aortic valve stenosis, hear murmurs, aortic valveprolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valveinsufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valveinsufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspidvalve insufficiency, and tricuspid valve stenosis.

Myocardial diseases include, but are not limited to, alcoholiccardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy,aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictivecardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis,endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury,and myocarditis.

Myocardial ischemias include, but are not limited to, coronary disease,such as angina pectoris, coronary aneurysm, coronary arteriosclerosis,coronary thrombosis, coronary vasospasm, myocardial infarction andmyocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

Aneurysms include, but are not limited to, dissecting aneurysms, falseaneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms,cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliacaneurysms.

Arterial occlusive diseases include, but are not limited to,arteriosclerosis, intermittent claudication, carotid stenosis,fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoyadisease, renal artery obstruction, retinal artery occlusion, andthromboangiitis obliterans.

Cerebrovascular disorders include, but are not limited to, carotidartery diseases, cerebral amyloid angiopathy, cerebral aneurysm,cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenousmalformation, cerebral artery diseases, cerebral embolism andthrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg'ssyndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma,subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia(including transient), subclavian steal syndrome, periventricularleukomalacia, vascular headache, cluster headache, migraine, andvertebrobasilar insufficiency.

Embolisms include, but are not limited to, air embolisms, amniotic fluidembolisms, cholesterol embolisms, blue toe syndrome, fat embolisms,pulmonary embolisms, and thromoboembolisms. Thrombosis include, but arenot limited to, coronary thrombosis, hepatic vein thrombosis, retinalvein occlusion, carotid artery thrombosis, sinus thrombosis,Wallenberg's syndrome, and thrombophlebitis.

Ischemic disorders include, but are not limited to, cerebral ischemia,ischemic colitis, compartment syndromes, anterior compartment syndrome,myocardial ischemia, reperfusion injuries, and peripheral limb ischemia.Vasculitis includes, but is not limited to, aortitis, arteritis,Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph nodesyndrome, thromboangiitis obliterans, hypersensitivity vasculitis,Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener'sgranulomatosis.

Polypeptides may be administered using any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,biolistic injectors, particle accelerators, gelfoam sponge depots, othercommercially available depot materials, osmotic pumps, oral orsuppositorial solid pharmaceutical formulations, decanting or topicalapplications during surgery, aerosol delivery. Such methods are known inthe art. Polypeptides may be administered as part of a Therapeutic,described in more detail below. Methods of delivering polynucleotidesare described in more detail herein.

Respiratory Disorders

Polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may be used to treat, prevent, diagnose, and/orprognose diseases and/or disorders of the respiratory system.

Diseases and disorders of the respiratory system include, but are notlimited to, nasal vestibulitis, nonallergic rhinitis (e.g., acuterhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis),nasal polyps, and sinusitis, juvenile angiofibromas, cancer of the noseand juvenile papillomas, vocal cord polyps, nodules (singer's nodules),contact ulcers, vocal cord paralysis, laryngoceles, pharyngitis (e.g.,viral and bacterial), tonsillitis, tonsillar cellulitis, parapharyngealabscess, laryngitis, laryngoceles, and throat cancers (e.g., cancer ofthe nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g.,squamous cell carcinoma, small cell (oat cell) carcinoma, large cellcarcinoma, and adenocarcinoma), allergic disorders (eosinophilicpneumonia, hypersensitivity pneumonitis (e.g., extrinsic allergicalveolitis, allergic interstitial pneumonitis, organic dustpneumoconiosis, allergic bronchopulmonary aspergillosis, asthma,Wegener's granulomatosis (granulomatous vasculitis), Goodpasture'ssyndrome)), pneumonia (e.g., bacterial pneumonia (e.g., Streptococcuspneumoniae (pneumoncoccal pneumonia), Staphylococcus aureus(staphylococcal pneumonia), Gram-negative bacterial pneumonia (causedby, e.g., Klebsiella and Pseudomas spp.), Mycoplasma pneumoniaepneumonia, Hemophilus influenzae pneumonia, Legionella pneumophila(Legionnaires' disease), and Chlamydia psittaci (Psittacosis)), andviral pneumonia (e.g., influenza, chickenpox (varicella).

Additional diseases and disorders of the respiratory system include, butare not limited to bronchiolitis, polio (poliomyelitis), croup,respiratory syncytial viral infection, mumps, erythema infectiosum(fifth disease), roseola infantum, progressive rubella panencephalitis,german measles, and subacute sclerosing panencephalitis), fungalpneumonia (e.g., Histoplasmosis, Coccidioidomycosis, Blastomycosis,fungal infections in people with severely suppressed immune systems(e.g., cryptococcosis, caused by Cryptococcus neoformans; aspergillosis,caused by Aspergillus spp.; candidiasis, caused by Candida; andmucormycosis)), Pneumocystis carinii (pneumocystis pneumonia), atypicalpneumonias (e.g., Mycoplasma and Chlamydia spp.), opportunisticinfection pneumonia, nosocomial pneumonia, chemical pneumonitis, andaspiration pneumonia, pleural disorders (e.g., pleurisy, pleuraleffusion, and pneumothorax (e.g., simple spontaneous pneumothorax,complicated spontaneous pneumothorax, tension pneumothorax)),obstructive airway diseases (e.g., asthma, chronic obstructive pulmonarydisease (COPD), emphysema, chronic or acute bronchitis), occupationallung diseases (e.g., silicosis, black lung (coal workers'pneumoconiosis), asbestosis, berylliosis, occupational asthsma,byssinosis, and benign pneumoconioses), Infiltrative Lung Disease (e.g.,pulmonary fibrosis (e.g., fibrosing alveolitis, usual interstitialpneumonia), idiopathic pulmonary fibrosis, desquamative interstitialpneumonia, lymphoid interstitial pneumonia, histiocytosis X (e.g.,Letterer-Siwe disease, Hand-Schüller-Christian disease, eosinophilicgranuloma), idiopathic pulmonary hemosiderosis, sarcoidosis andpulmonary alveolar proteinosis), Acute respiratory distress syndrome(also called, e.g., adult respiratory distress syndrome), edema,pulmonary embolism, bronchitis (e.g., viral, bacterial), bronchiectasis,atelectasis, lung abscess (caused by, e.g., Staphylococcus aureus orLegionella pneumophila), and cystic fibrosis.

Anti-Angiogenesis Activity

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

The present invention provides for treatment of diseases or disordersassociated with neovascularization by administration of thepolynucleotides and/or polypeptides of the invention, as well asagonists or antagonists of the present invention. Malignant andmetastatic conditions which can be treated with the polynucleotides andpolypeptides, or agonists or antagonists of the invention include, butare not limited to, malignancies, solid tumors, and cancers describedherein and otherwise known in the art (for a review of such disorders,see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia(1985)). Thus, the present invention provides a method of treating anangiogenesis-related disease and/or disorder, comprising administeringto an individual in need thereof a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist of the invention.For example, polynucleotides, polypeptides, antagonists and/or agonistsmay be utilized in a variety of additional methods in order totherapeutically treat a cancer or tumor. Cancers which may be treatedwith polynucleotides, polypeptides, antagonists and/or agonists include,but are not limited to solid tumors, including prostate, lung, breast,ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid,biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,bladder, thyroid cancer; primary tumors and metastases; melanomas;glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lungcancer; colorectal cancer; advanced malignancies; and blood born tumorssuch as leukemias. For example, polynucleotides, polypeptides,antagonists and/or agonists may be delivered topically, in order totreat cancers such as skin cancer, head and neck tumors, breast tumors,and Kaposi's sarcoma.

Within yet other aspects, polynucleotides, polypeptides, antagonistsand/or agonists may be utilized to treat superficial forms of bladdercancer by, for example, intravesical administration. Polynucleotides,polypeptides, antagonists and/or agonists may be delivered directly intothe tumor, or near the tumor site, via injection or a catheter. Ofcourse, as the artisan of ordinary skill will appreciate, theappropriate mode of administration will vary according to the cancer tobe treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and/or agonists may be usefulin treating other disorders, besides cancers, which involveangiogenesis. These disorders include, but are not limited to: benigntumors, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas; artheroscleric plaques; ocularangiogenic diseases, for example, diabetic retinopathy, retinopathy ofprematurity, macular degeneration, corneal graft rejection, neovascularglaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis andPterygia (abnormal blood vessel growth) of the eye; rheumatoidarthritis; psoriasis; delayed wound healing; endometriosis;vasculogenesis; granulations; hypertrophic scars (keloids); nonunionfractures; scleroderma; trachoma; vascular adhesions; myocardialangiogenesis; coronary collaterals; cerebral collaterals; arteriovenousmalformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;fibromuscular dysplasia; wound granulation; Crohn's disease; andatherosclerosis.

For example, within one aspect of the present invention methods areprovided for treating hypertrophic scars and keloids, comprising thestep of administering a polynucleotide, polypeptide, antagonist and/oragonist of the invention to a hypertrophic scar or keloid.

Within one embodiment of the present invention polynucleotides,polypeptides, antagonists and/or agonists of the invention are directlyinjected into a hypertrophic scar or keloid, in order to prevent theprogression of these lesions. This therapy is of particular value in theprophylactic treatment of conditions which are known to result in thedevelopment of hypertrophic scars and keloids (e.g., burns), and ispreferably initiated after the proliferative phase has had time toprogress (approximately 14 days after the initial injury), but beforehypertrophic scar or keloid development. As noted above, the presentinvention also provides methods for treating neovascular diseases of theeye, including for example, corneal neovascularization, neovascularglaucoma, proliferative diabetic retinopathy, retrolental fibroplasiaand macular degeneration.

Moreover, Ocular disorders associated with neovascularization which canbe treated with the polynucleotides and polypeptides of the presentinvention (including agonists and/or antagonists) include, but are notlimited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma,retrolental fibroplasia, uveitis, retinopathy of prematurity maculardegeneration, corneal graft neovascularization, as well as other eyeinflammatory diseases, ocular tumors and diseases associated withchoroidal or iris neovascularization. See, e.g., reviews by Waltman etal., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al., Surv.Ophthal. 22:291-312 (1978).

Thus, within one aspect of the present invention methods are providedfor treating neovascular diseases of the eye such as cornealneovascularization (including corneal graft neovascularization),comprising the step of administering to a patient a therapeuticallyeffective amount of a compound (as described above) to the cornea, suchthat the formation of blood vessels is inhibited. Briefly, the cornea isa tissue which normally lacks blood vessels. In certain pathologicalconditions however, capillaries may extend into the cornea from thepericorneal vascular plexus of the limbus. When the cornea becomesvascularized, it also becomes clouded, resulting in a decline in thepatient's visual acuity. Visual loss may become complete if the corneacompletely opacitates. A wide variety of disorders can result in cornealneovascularization, including for example, corneal infections (e.g.,trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis),immunological processes (e.g., graft rejection and Stevens-Johnson'ssyndrome), alkali burns, trauma, inflammation (of any cause), toxic andnutritional deficiency states, and as a complication of wearing contactlenses.

Within particularly preferred embodiments of the invention, may beprepared for topical administration in saline (combined with any of thepreservatives and antimicrobial agents commonly used in ocularpreparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily. Alternatively, anti-angiogenic compositions, prepared asdescribed above, may also be administered directly to the cornea. Withinpreferred embodiments, the anti-angiogenic composition is prepared witha muco-adhesive polymer which binds to cornea. Within furtherembodiments, the anti-angiogenic factors or anti-angiogenic compositionsmay be utilized as an adjunct to conventional steroid therapy. Topicaltherapy may also be useful prophylactically in corneal lesions which areknown to have a high probability of inducing an angiogenic response(such as chemical burns). In these instances the treatment, likely incombination with steroids, may be instituted immediately to help preventsubsequent complications.

Within other embodiments, the compounds described above may be injecteddirectly into the corneal stroma by an ophthalmologist under microscopicguidance. The preferred site of injection may vary with the morphologyof the individual lesion, but the goal of the administration would be toplace the composition at the advancing front of the vasculature (i.e.,interspersed between the blood vessels and the normal cornea). In mostcases this would involve perilimbic corneal injection to “protect” thecornea from the advancing blood vessels. This method may also beutilized shortly after a corneal insult in order to prophylacticallyprevent corneal neovascularization. In this situation the material couldbe injected in the perilimbic cornea interspersed between the corneallesion and its undesired potential limbic blood supply. Such methods mayalso be utilized in a similar fashion to prevent capillary invasion oftransplanted corneas. In a sustained-release form injections might onlybe required 2-3 times per year. A steroid could also be added to theinjection solution to reduce inflammation resulting from the injectionitself.

Within another aspect of the present invention, methods are provided fortreating neovascular glaucoma, comprising the step of administering to apatient a therapeutically effective amount of a polynucleotide,polypeptide, antagonist and/or agonist to the eye, such that theformation of blood vessels is inhibited. In one embodiment, the compoundmay be administered topically to the eye in order to treat early formsof neovascular glaucoma. Within other embodiments, the compound may beimplanted by injection into the region of the anterior chamber angle.Within other embodiments, the compound may also be placed in anylocation such that the compound is continuously released into theaqueous humor. Within another aspect of the present invention, methodsare provided for treating proliferative diabetic retinopathy, comprisingthe step of administering to a patient a therapeutically effectiveamount of a polynucleotide, polypeptide, antagonist and/or agonist tothe eyes, such that the formation of blood vessels is inhibited.

Within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection into theaqueous humor or the vitreous, in order to increase the localconcentration of the polynucleotide, polypeptide, antagonist and/oragonist in the retina. Preferably, this treatment should be initiatedprior to the acquisition of severe disease requiring photocoagulation.

Within another aspect of the present invention, methods are provided fortreating retrolental fibroplasia, comprising the step of administeringto a patient a therapeutically effective amount of a polynucleotide,polypeptide, antagonist and/or agonist to the eye, such that theformation of blood vessels is inhibited. The compound may beadministered topically, via intravitreous injection and/or viaintraocular implants.

Additionally, disorders which can be treated with the polynucleotides,polypeptides, agonists and/or agonists include, but are not limited to,hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques,delayed wound healing, granulations, hemophilic joints, hypertrophicscars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

Moreover, disorders and/or states, which can be treated, prevented,diagnosed, and/or prognosed with the the polynucleotides, polypeptides,agonists and/or agonists of the invention include, but are not limitedto, solid tumors, blood born tumors such as leukemias, tumor metastasis,Kaposi's sarcoma, benign tumors, for example hemangiomas, acousticneuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoidarthritis, psoriasis, ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis, retinoblastoma, and uvietis, delayed wound healing,endometriosis, vascluogenesis, granulations, hypertrophic scars(keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

In one aspect of the birth control method, an amount of the compoundsufficient to block embryo implantation is administered before or afterintercourse and fertilization have occurred, thus providing an effectivemethod of birth control, possibly a “morning after” method.Polynucleotides, polypeptides, agonists and/or agonists may also be usedin controlling menstruation or administered as either a peritoneallavage fluid or for peritoneal implantation in the treatment ofendometriosis.

Polynucleotides, polypeptides, agonists and/or agonists of the presentinvention may be incorporated into surgical sutures in order to preventstitch granulomas.

Polynucleotides, polypeptides, agonists and/or agonists may be utilizedin a wide variety of surgical procedures. For example, within one aspectof the present invention a compositions (in the form of, for example, aspray or film) may be utilized to coat or spray an area prior to removalof a tumor, in order to isolate normal surrounding tissues frommalignant tissue, and/or to prevent the spread of disease to surroundingtissues. Within other aspects of the present invention, compositions(e.g., in the form of a spray) may be delivered via endoscopicprocedures in order to coat tumors, or inhibit angiogenesis in a desiredlocale. Within yet other aspects of the present invention, surgicalmeshes which have been coated with anti-angiogenic compositions of thepresent invention may be utilized in any procedure wherein a surgicalmesh might be utilized. For example, within one embodiment of theinvention a surgical mesh laden with an anti-angiogenic composition maybe utilized during abdominal cancer resection surgery (e.g., subsequentto colon resection) in order to provide support to the structure, and torelease an amount of the anti-angiogenic factor.

Within further aspects of the present invention, methods are providedfor treating tumor excision sites, comprising administering apolynucleotide, polypeptide, agonist and/or agonist to the resectionmargins of a tumor subsequent to excision, such that the localrecurrence of cancer and the formation of new blood vessels at the siteis inhibited. Within one embodiment of the invention, theanti-angiogenic compound is administered directly to the tumor excisionsite (e.g., applied by swabbing, brushing or otherwise coating theresection margins of the tumor with the anti-angiogenic compound).Alternatively, the anti-angiogenic compounds may be incorporated intoknown surgical pastes prior to administration. Within particularlypreferred embodiments of the invention, the anti-angiogenic compoundsare applied after hepatic resections for malignancy, and afterneurosurgical operations.

Within one aspect of the present invention, polynucleotides,polypeptides, agonists and/or agonists may be administered to theresection margin of a wide variety of tumors, including for example,breast, colon, brain and hepatic tumors. For example, within oneembodiment of the invention, anti-angiogenic compounds may beadministered to the site of a neurological tumor subsequent to excision,such that the formation of new blood vessels at the site are inhibited.

The polynucleotides, polypeptides, agonists and/or agonists of thepresent invention may also be administered along with otheranti-angiogenic factors. Representative examples of otheranti-angiogenic factors include: Anti-Invasive Factor, retinoic acid andderivatives thereof, paclitaxel, Suramin, Tissue Inhibitor ofMetalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2,and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude platelet factor 4; protamine sulphate; sulphated chitinderivatives (prepared from queen crab shells), (Murata et al., CancerRes. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex(SP-PG) (the function of this compound may be enhanced by the presenceof steroids such as estrogen, and tamoxifen citrate); Staurosporine;modulators of matrix metabolism, including for example, proline analogs,cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;Angostatic steroid; AGM-1470; carboxynaminolmidazole; andmetalloproteinase inhibitors such as BB94.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition ofapoptosis that could be treated, prevented, diagnosed, and/or prognosedusing polynucleotides or polypeptides, as well as antagonists oragonists of the present invention, include cancers (such as follicularlymphomas, carcinomas with p53 mutations, and hormone-dependent tumors,including, but not limited to colon cancer, cardiac tumors, pancreaticcancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinalcancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection.

In preferred embodiments, polynucleotides, polypeptides, and/orantagonists of the invention are used to inhibit growth, progression,and/or metasis of cancers, in particular those listed above.

Additional diseases or conditions associated with increased cellsurvival that could be treated or detected by polynucleotides orpolypeptides, or agonists or antagonists of the present inventioninclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with increased apoptosis that could be treated,prevented, diagnosed, and/or prognesed using polynucleotides orpolypeptides, as well as agonists or antagonists of the presentinvention, include, but are not limited to, AIDS; neurodegenerativedisorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophiclateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration andbrain tumor or prior associated disease); autoimmune disorders (such as,multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) myelodysplastic syndromes (such as aplastic anemia), graft v.host disease, ischemic injury (such as that caused by myocardialinfarction, stroke and reperfusion injury), liver injury (e.g.,hepatitis related liver injury, ischemia/reperfusion injury, cholestosis(bile duct injury) and liver cancer); toxin-induced liver disease (suchas that caused by alcohol), septic shock, cachexia and anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing polynucleotides or polypeptides, aswell as agonists or antagonists of the present invention, fortherapeutic purposes, for example, to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of wound healing,and to stimulate hair follicle production and healing of dermal wounds.Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, may be clinically useful in stimulating woundhealing including surgical wounds, excisional wounds, deep woundsinvolving damage of the dermis and epidermis, eye tissue wounds, dentaltissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers,cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resultingfrom heat exposure or chemicals, and other abnormal wound healingconditions such as uremia, malnutrition, vitamin deficiencies andcomplications associated with systemic treatment with steroids,radiation therapy and antineoplastic drugs and antimetabolites.Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could be used to promote dermal reestablishmentsubsequent to dermal loss

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could be used to increase the adherence of skingrafts to a wound bed and to stimulate re-epithelialization from thewound bed. The following are types of grafts that polynucleotides orpolypeptides, agonists or antagonists of the present invention, could beused to increase adherence to a wound bed: autografts, artificial skin,allografts, autodermic graft, autoepdermic grafts, avacular grafts,Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft,delayed graft, dermic graft, epidermic graft, fascia graft, fullthickness graft, heterologous graft, xenograft, homologous graft,hyperplastic graft, lamellar graft, mesh graft, mucosal graft,Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,penetrating graft, split skin graft, thick split graft. Polynucleotidesor polypeptides, as well as agonists or antagonists of the presentinvention, can be used to promote skin strength and to improve theappearance of aged skin.

It is believed that polynucleotides or polypeptides, as well as agonistsor antagonists of the present invention, will also produce changes inhepatocyte proliferation, and epithelial cell proliferation in the lung,breast, pancreas, stomach, small intestine, and large intestine.Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could promote proliferation of epithelial cellssuch as sebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. Polynucleotides or polypeptides, agonists or antagonists of thepresent invention, may promote proliferation of endothelial cells,keratinocytes, and basal keratinocytes.

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could also be used to reduce the side effects ofgut toxicity that result from radiation, chemotherapy treatments orviral infections. Polynucleotides or polypeptides, as well as agonistsor antagonists of the present invention, may have a cytoprotectiveeffect on the small intestine mucosa. Polynucleotides or polypeptides,as well as agonists or antagonists of the present invention, may alsostimulate healing of mucositis (mouth ulcers) that result fromchemotherapy and viral infections.

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could further be used in full regeneration ofskin in full and partial thickness skin defects, including burns, (i.e.,repopulation of hair follicles, sweat glands, and sebaceous glands),treatment of other skin defects such as psoriasis. Polynucleotides orpolypeptides, as well as agonists or antagonists of the presentinvention, could be used to treat epidermolysis bullosa, a defect inadherence of the epidermis to the underlying dermis which results infrequent, open and painful blisters by accelerating reepithelializationof these lesions. Polynucleotides or polypeptides, as well as agonistsor antagonists of the present invention, could also be used to treatgastric and doudenal ulcers and help heal by scar formation of themucosal lining and regeneration of glandular mucosa and duodenal mucosallining more rapidly. Inflammatory bowel diseases, such as Crohn'sdisease and ulcerative colitis, are diseases which result in destructionof the mucosal surface of the small or large intestine, respectively.Thus, polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could be used to promote theresurfacing of the mucosal surface to aid more rapid healing and toprevent progression of inflammatory bowel disease. Treatment withpolynucleotides or polypeptides, agonists or antagonists of the presentinvention, is expected to have a significant effect on the production ofmucus throughout the gastrointestinal tract and could be used to protectthe intestinal mucosa from injurious substances that are ingested orfollowing surgery. Polynucleotides or polypeptides, as well as agonistsor antagonists of the present invention, could be used to treat diseasesassociate with the under expression.

Moreover, polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could be used to prevent and healdamage to the lungs due to various pathological states. Polynucleotidesor polypeptides, as well as agonists or antagonists of the presentinvention, which could stimulate proliferation and differentiation andpromote the repair of alveoli and brochiolar epithelium to prevent ortreat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of aveoli, and inhalation injuries,i.e., resulting from smoke inhalation and burns, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treatedusing polynucleotides or polypeptides, agonists or antagonists of thepresent invention. Also, polynucleotides or polypeptides, as well asagonists or antagonists of the present invention, could be used tostimulate the proliferation of and differentiation of type IIpneumocytes, which may help treat or prevent disease such as hyalinemembrane diseases, such as infant respiratory distress syndrome andbronchopulmonary displasia, in premature infants.

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, could stimulate the proliferation anddifferentiation of hepatocytes and, thus, could be used to alleviate ortreat liver diseases and pathologies such as fulminant liver failurecaused by cirrhosis, liver damage caused by viral hepatitis and toxicsubstances (i.e., acetaminophen, carbon tetraholoride and otherhepatotoxins known in the art).

In addition, polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could be used treat or prevent theonset of diabetes mellitus. In patients with newly diagnosed Types I andII diabetes, where some islet cell function remains, polynucleotides orpolypeptides, as well as agonists or antagonists of the presentinvention, could be used to maintain the islet function so as toalleviate, delay or prevent permanent manifestation of the disease.Also, polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, could be used as an auxiliary inislet cell transplantation to improve or promote islet cell function.

Neural Activity and Neurological Diseases

The polynucleotides, polypeptides and agonists or antagonists of theinvention may be used for the diagnosis and/or treatment of diseases,disorders, damage or injury of the brain and/or nervous system. Nervoussystem disorders that can be treated with the compositions of theinvention (e.g., polypeptides, polynucleotides, and/or agonists orantagonists), include, but are not limited to, nervous system injuries,and diseases or disorders which result in either a disconnection ofaxons, a diminution or degeneration of neurons, or demyelination.Nervous system lesions which may be treated in a patient (includinghuman and non-human mammalian patients) according to the methods of theinvention, include but are not limited to, the following lesions ofeither the central (including spinal cord, brain) or peripheral nervoussystems: (1) ischemic lesions, in which a lack of oxygen in a portion ofthe nervous system results in neuronal injury or death, includingcerebral infarction or ischemia, or spinal cord infarction or ischemia;(2) traumatic lesions, including lesions caused by physical injury orassociated with surgery, for example, lesions which sever a portion ofthe nervous system, or compression injuries; (3) malignant lesions, inwhich a portion of the nervous system is destroyed or injured bymalignant tissue which is either a nervous system associated malignancyor a malignancy derived from non-nervous system tissue; (4) infectiouslesions, in which a portion of the nervous system is destroyed orinjured as a result of infection, for example, by an abscess orassociated with infection by human immunodeficiency virus, herpeszoster, or herpes simplex virus or with Lyme disease, tuberculosis, orsyphilis; (5) degenerative lesions, in which a portion of the nervoussystem is destroyed or injured as a result of a degenerative processincluding but not limited to, degeneration associated with Parkinson'sdisease, Alzheimer's disease, Huntington's chorea, or amyotrophiclateral sclerosis (ALS); (6) lesions associated with nutritionaldiseases or disorders, in which a portion of the nervous system isdestroyed or injured by a nutritional disorder or disorder of metabolismincluding, but not limited to, vitamin B12 deficiency, folic aciddeficiency, Wernicke disease, tobacco-alcohol amblyopia,Marchiafava-Bignami disease (primary degeneration of the corpuscallosum), and alcoholic cerebellar degeneration; (7) neurologicallesions associated with systemic diseases including, but not limited to,diabetes (diabetic neuropathy, Bell's palsy), systemic lupuserythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxicsubstances including alcohol, lead, or particular neurotoxins; and (9)demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

In one embodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to protect neural cells from thedamaging effects of hypoxia. In a further preferred embodiment, thepolypeptides, polynucleotides, or agonists or antagonists of theinvention are used to protect neural cells from the damaging effects ofcerebral hypoxia. According to this embodiment, the compositions of theinvention are used to treat or prevent neural cell injury associatedwith cerebral hypoxia. In one non-exclusive aspect of this embodiment,the polypeptides, polynucleotides, or agonists or antagonists of theinvention, are used to treat or prevent neural cell injury associatedwith cerebral ischemia. In another non-exclusive aspect of thisembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat or prevent neural cellinjury associated with cerebral infarction.

In another preferred embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat or preventneural cell injury associated with a stroke. In a specific embodiment,the polypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat or prevent cerebral neural cell injuryassociated with a stroke.

In another preferred embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat or preventneural cell injury associated with a heart attack. In a specificembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat or prevent cerebralneural cell injury associated with a heart attack.

The compositions of the invention which are useful for treating orpreventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture either in the presence or absence of hypoxia or hypoxicconditions; (2) increased sprouting of neurons in culture or in vivo;(3) increased production of a neuron-associated molecule in culture orin vivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, in Zhang et al., ProcNatl Acad Sci USA 97:3637-42 (2000) or in Arakawa et al., J. Neurosci.,10:3507-15 (1990); increased sprouting of neurons may be detected bymethods known in the art, such as, for example, the methods set forth inPestronk et al., Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann.Rev. Neurosci., 4:17-42 (1981); increased production ofneuron-associated molecules may be measured by bioassay, enzymaticassay, antibody binding, Northern blot assay, etc., using techniquesknown in the art and depending on the molecule to be measured; and motorneuron dysfunction may be measured by assessing the physicalmanifestation of motor neuron disorder, e.g., weakness, motor neuronconduction velocity, or functional disability.

In specific embodiments, motor neuron disorders that may be treatedaccording to the invention include, but are not limited to, disorderssuch as infarction, infection, exposure to toxin, trauma, surgicaldamage, degenerative disease or malignancy that may affect motor neuronsas well as other components of the nervous system, as well as disordersthat selectively affect neurons such as amyotrophic lateral sclerosis,and including, but not limited to, progressive spinal muscular atrophy,progressive bulbar palsy, primary lateral sclerosis, infantile andjuvenile muscular atrophy, progressive bulbar paralysis of childhood(Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, andHereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

Further, polypeptides or polynucleotides of the invention may play arole in neuronal survival; synapse formation; conductance; neuraldifferentiation, etc. Thus, compositions of the invention (includingpolynucleotides, polypeptides, and agonists or antagonists) may be usedto diagnose and/or treat or prevent diseases or disorders associatedwith these roles, including, but not limited to, learning and/orcognition disorders. The compositions of the invention may also beuseful in the treatment or prevention of neurodegenerative diseasestates and/or behavioural disorders. Such neurodegenerative diseasestates and/or behavioral disorders include, but are not limited to,Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, TouretteSyndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsivedisorder, panic disorder, learning disabilities, ALS, psychoses, autism,and altered behaviors, including disorders in feeding, sleep patterns,balance, and perception. In addition, compositions of the invention mayalso play a role in the treatment, prevention and/or detection ofdevelopmental disorders associated with the developing embryo, orsexually-linked disorders.

Additionally, polypeptides, polynucleotides and/or agonists orantagonists of the invention, may be useful in protecting neural cellsfrom diseases, damage, disorders, or injury, associated withcerebrovascular disorders including, but not limited to, carotid arterydiseases (e.g., carotid artery thrombosis, carotid stenosis, or MoyamoyaDisease), cerebral amyloid angiopathy, cerebral aneurysm, cerebralanoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations,cerebral artery diseases, cerebral embolism and thrombosis (e.g.,carotid artery thrombosis, sinus thrombosis, or Wallenberg's Syndrome),cerebral hemorrhage (e.g., epidural or subdural hematoma, orsubarachnoid hemorrhage), cerebral infarction, cerebral ischemia (e.g.,transient cerebral ischemia, Subclavian Steal Syndrome, orvertebrobasilar insufficiency), vascular dementia (e.g., multi-infarct),leukomalacia, periventricular, and vascular headache (e.g., clusterheadache or migraines).

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing polynucleotides or polypeptides, aswell as agonists or antagonists of the present invention, fortherapeutic purposes, for example, to stimulate neurological cellproliferation and/or differentiation. Therefore, polynucleotides,polypeptides, agonists and/or antagonists of the invention may be usedto treat and/or detect neurologic diseases. Moreover, polynucleotides orpolypeptides, or agonists or antagonists of the invention, can be usedas a marker or detector of a particular nervous system disease ordisorder.

Examples of neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include brain diseases, such as metabolic braindiseases which includes phenylketonuria such as maternalphenylketonuria, pyruvate carboxylase deficiency, pyruvate dehydrogenasecomplex deficiency, Wernicke's Encephalopathy, brain edema, brainneoplasms such as cerebellar neoplasms which include infratentorialneoplasms, cerebral ventricle neoplasms such as choroid plexusneoplasms, hypothalamic neoplasms, supratentorial neoplasms, canavandisease, cerebellar diseases such as cerebellar ataxia which includespinocerebellar degeneration such as ataxia telangiectasia, cerebellardyssynergia, Friederich's Ataxia, Machado-Joseph Disease,olivopontocerebellar atrophy, cerebellar neoplasms such asinfratentorial neoplasms, diffuse cerebral sclerosis such asencephalitis periaxialis, globoid cell leukodystrophy, metachromaticleukodystrophy and subacute sclerosing panencephalitis.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include cerebrovascular disorders (such as carotidartery diseases which include carotid artery thrombosis, carotidstenosis and Moyamoya Disease), cerebral amyloid angiopathy, cerebralaneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebralarteriovenous malformations, cerebral artery diseases, cerebral embolismand thrombosis such as carotid artery thrombosis, sinus thrombosis andWallenberg's Syndrome, cerebral hemorrhage such as epidural hematoma,subdural hematoma and subarachnoid hemorrhage, cerebral infarction,cerebral ischemia such as transient cerebral ischemia, Subclavian StealSyndrome and vertebrobasilar insufficiency, vascular dementia such asmulti-infarct dementia, periventricular leukomalacia, vascular headachesuch as cluster headache and migraine.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include dementia such as AIDS Dementia Complex,presenile dementia such as Alzheimer's Disease and Creutzfeldt-JakobSyndrome, senile dementia such as Alzheimer's Disease and progressivesupranuclear palsy, vascular dementia such as multi-infarct dementia,encephalitis which include encephalitis periaxialis, viral encephalitissuch as epidemic encephalitis, Japanese Encephalitis, St. LouisEncephalitis, tick-borne encephalitis and West Nile Fever, acutedisseminated encephalomyelitis, meningoencephalitis such asuveomeningoencephalitic syndrome, Postencephalitic Parkinson Disease andsubacute sclerosing panencephalitis, encephalomalacia such asperiventricular leukomalacia, epilepsy such as generalized epilepsywhich includes infantile spasms, absence epilepsy, myoclonic epilepsywhich includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsysuch as complex partial epilepsy, frontal lobe epilepsy and temporallobe epilepsy, post-traumatic epilepsy, status epilepticus such asEpilepsia Partialis Continua, and Hallervorden-Spatz Syndrome.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include hydrocephalus such as Dandy-Walker Syndromeand normal pressure hydrocephalus, hypothalamic diseases such ashypothalamic neoplasms, cerebral malaria, narcolepsy which includescataplexy, bulbar poliomyelitis, cerebri pseudotumor, Rett Syndrome,Reye's Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranialtuberculoma and Zellweger Syndrome, central nervous system infectionssuch as AIDS Dementia Complex, Brain Abscess, subdural empyema,encephalomyelitis such as Equine Encephalomyelitis, Venezuelan EquineEncephalomyelitis, Necrotizing Hemorrhagic Encephalomyelitis, Visna, andcerebral malaria.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include meningitis such as arachnoiditis, asepticmeningtitis such as viral meningtitis which includes lymphocyticchoriomeningitis, Bacterial meningtitis which includes HaemophilusMeningtitis, Listeria Meningtitis, Meningococcal Meningtitis such asWaterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningealtuberculosis, fungal meningitis such as Cryptococcal Meningtitis,subdural effusion, meningoencephalitis such as uvemeningoencephaliticsyndrome, myelitis such as transverse myelitis, neurosyphilis such astabes dorsalis, poliomyelitis which includes bulbar poliomyelitis andpostpoliomyelitis syndrome, prion diseases (such as Creutzfeldt-JakobSyndrome, Bovine Spongiform Encephalopathy, Gerstmann-StrausslerSyndrome, Kuru, Scrapie), and cerebral toxoplasmosis.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include central nervous system neoplasms such as brainneoplasms that include cerebellar neoplasms such as infratentorialneoplasms, cerebral ventricle neoplasms such as choroid plexusneoplasms, hypothalamic neoplasms and supratentorial neoplasms,meningeal neoplasms, spinal cord neoplasms which include epiduralneoplasms, demyelinating diseases such as Canavan Diseases, diffusecerebral sceloris which includes adrenoleukodystrophy, encephalitisperiaxialis, globoid cell leukodystrophy, diffuse cerebral sclerosissuch as metachromatic leukodystrophy, allergic encephalomyelitis,necrotizing hemorrhagic encephalomyelitis, progressive multifocalleukoencephalopathy, multiple sclerosis, central pontine myelinolysis,transverse myelitis, neuromyelitis optica, Scrapie, Swayback, ChronicFatigue Syndrome, Visna, High Pressure Nervous Syndrome, Meningism,spinal cord diseases such as amyotonia congenita, amyotrophic lateralsclerosis, spinal muscular atrophy such as Werdnig-Hoffmann Disease,spinal cord compression, spinal cord neoplasms such as epiduralneoplasms, syringomyelia, Tabes Dorsalis, Stiff-Man Syndrome, mentalretardation such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange'sSyndrome, Down Syndrome, Gangliosidoses such as gangliosidoses G(M1),Sandhoff Disease, Tay-Sachs Disease, Hartnup Disease, homocystinuria,Laurence-Moon-Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup UrineDisease, mucolipidosis such as fucosidosis, neuronalceroid-lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria suchas maternal phenylketonuria, Prader-Willi Syndrome, Rett Syndrome,Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome, nervoussystem abnormalities such as holoprosencephaly, neural tube defects suchas anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity,encephalocele, meningocele, meningomyelocele, spinal dysraphism such asspina bifida cystica and spina bifida occulta.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include hereditary motor and sensory neuropathieswhich include Charcot-Marie Disease, Hereditary optic atrophy, Refsum'sDisease, hereditary spastic paraplegia, Werdnig-Hoffmann Disease,Hereditary Sensory and Autonomic Neuropathies such as CongenitalAnalgesia and Familial Dysautonomia, Neurologic manifestations (such asagnosia that include Gerstmann's Syndrome, Amnesia such as retrogradeamnesia, apraxia, neurogenic bladder, cataplexy, communicative disorderssuch as hearing disorders that includes deafness, partial hearing loss,loudness recruitment and tinnitus, language disorders such as aphasiawhich include agraphia, anomia, broca aphasia, and Wernicke Aphasia,Dyslexia such as Acquired Dyslexia, language development disorders,speech disorders such as aphasia which includes anomia, broca aphasiaand Wernicke Aphasia, articulation disorders, communicative disorderssuch as speech disorders which include dysarthria, echolalia, mutism andstuttering, voice disorders such as aphonia and hoarseness, decerebratestate, delirium, fasciculation, hallucinations, meningism, movementdisorders such as angelman syndrome, ataxia, athetosis, chorea,dystonia, hypokinesia, muscle hypotonia, myoclonus, tic, torticollis andtremor, muscle hypertonia such as muscle rigidity such as stiff-mansyndrome, muscle spasticity, paralysis such as facial paralysis whichincludes Herpes Zoster Oticus, Gastroparesis, Hemiplegia,ophthalmoplegia such as diplopia, Duane's Syndrome, Horner's Syndrome,Chronic progressive external ophthalmoplegia such as Kearns Syndrome,Bulbar Paralysis, Tropical Spastic Paraparesis, Paraplegia such asBrown-Sequard Syndrome, quadriplegia, respiratory paralysis and vocalcord paralysis, paresis, phantom limb, taste disorders such as ageusiaand dysgeusia, vision disorders such as amblyopia, blindness, colorvision defects, diplopia, hemianopsia, scotoma and subnormal vision,sleep disorders such as hypersomnia which includes Kleine-LevinSyndrome, insomnia, and somnambulism, spasm such as trismus,unconsciousness such as coma, persistent vegetative state and syncopeand vertigo, neuromuscular diseases such as amyotonia congenita,amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motorneuron disease, muscular atrophy such as spinal muscular atrophy,Charcot-Marie Disease and Werdnig-Hoffmann Disease, PostpoliomyelitisSyndrome, Muscular Dystrophy, Myasthenia Gravis, Myotonia Atrophica,Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis,Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff-ManSyndrome, peripheral nervous system diseases such as acrodynia, amyloidneuropathies, autonomic nervous system diseases such as Adie's Syndrome,Barre-Lieou Syndrome, Familial Dysautonomia, Homer's Syndrome, ReflexSympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseasessuch as Acoustic Nerve Diseases such as Acoustic Neuroma which includesNeurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia,Melkersson-Rosenthal Syndrome, ocular motility disorders which includesamblyopia, nystagmus, oculomotor nerve paralysis, ophthalmoplegia suchas Duane's Syndrome, Homer's Syndrome, Chronic Progressive ExternalOphthalmoplegia which includes Kearns Syndrome, Strabismus such asEsotropia and Exotropia, Oculomotor Nerve Paralysis, Optic NerveDiseases such as Optic Atrophy which includes Hereditary Optic Atrophy,Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica,Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, DemyelinatingDiseases such as Neuromyelitis Optica-and Swayback, and Diabeticneuropathies such as diabetic foot.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include nerve compression syndromes such as carpaltunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome suchas cervical rib syndrome, ulnar nerve compression syndrome, neuralgiasuch as causalgia, cervico-brachial neuralgia, facial neuralgia andtrigeminal neuralgia, neuritis such as experimental allergic neuritis,optic neuritis, polyneuritis, polyradiculoneuritis and radiculities suchas polyradiculitis, hereditary motor and sensory neuropathies such asCharcot-Marie Disease, Hereditary Optic Atrophy, Refsum's Disease,Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, HereditarySensory and Autonomic Neuropathies which include Congenital Analgesiaand Familial Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweatingand Tetany).

Endocrine Disorders

Polynucleotides or polypeptides, or agonists or antagonists of thepresent invention, may be used to treat, prevent, diagnose, and/orprognose disorders and/or diseases related to hormone imbalance, and/ordisorders or diseases of the endocrine system.

Hormones secreted by the glands of the endocrine system control physicalgrowth, sexual function, metabolism, and other functions. Disorders maybe classified in two ways: disturbances in the production of hormones,and the inability of tissues to respond to hormones. The etiology ofthese hormone imbalance or endocrine system diseases, disorders orconditions may be genetic, somatic, such as cancer and some autoimmunediseases, acquired (e.g., by chemotherapy, injury or toxins), orinfectious. Moreover, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention can be used as a markeror detector of a particular disease or disorder related to the endocrinesystem and/or hormone imbalance.

Endocrine system and/or hormone imbalance and/or diseases encompassdisorders of uterine motility including, but not limited to:complications with pregnancy and labor (e.g., pre-term labor, post-termpregnancy, spontaneous abortion, and slow or stopped labor); anddisorders and/or diseases of the menstrual cycle (e.g., dysmenorrhea andendometriosis).

Endocrine system and/or hormone imbalance disorders and/or diseasesinclude disorders and/or diseases of the pancreas, such as, for example,diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis,pheochromocytoma—islet cell tumor syndrome; disorders and/or diseases ofthe adrenal glands such as, for example, Addison's Disease,corticosteroid deficiency, virilizing disease, hirsutism, Cushing'sSyndrome, hyperaldosteronism, pheochromocytoma; disorders and/ordiseases of the pituitary gland, such as, for example, hyperpituitarism,hypopituitarism, pituitary dwarfism, pituitary adenoma,panhypopituitarism, acromegaly, gigantism; disorders and/or diseases ofthe thyroid, including but not limited to, hyperthyroidism,hypothyroidism, Plummer's disease, Graves' disease (toxic diffusegoiter), toxic nodular goiter, thyroiditis (Hashimoto's thyroiditis,subacute granulomatous thyroiditis, and silent lymphocytic thyroiditis),Pendred's syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormonecoupling defect, thymic aplasia, Hurthle cell tumours of the thyroid,thyroid cancer, thyroid carcinoma, Medullary thyroid carcinoma;disorders and/or diseases of the parathyroid, such as, for example,hyperparathyroidism, hypoparathyroidism; disorders and/or diseases ofthe hypothalamus.

In specific embodiments, the polynucleotides and/or polypeptidescorresponding to this gene and/or agonists or antagonists of thosepolypeptides (including antibodies) as well as fragments and variants ofthose polynucleotides, polypeptides, agonists and antagonists, may beused to diagnose, prognose, treat, prevent, or ameliorate diseases anddisorders associated with aberrant glucose metabolism or glucose uptakeinto cells.

In a specific embodiment, the polynucleotides and/or polypeptidescorresponding to this gene and/or agonists and/or antagonists thereofmay be used to diagnose, prognose, treat, prevent, and/or amelioratetype I diabetes mellitus (insulin dependent diabetes mellitus, IDDM).

In another embodiment, the polynucleotides and/or polypeptidescorresponding to this gene and/or agonists and/or antagonists thereofmay be used to diagnose, prognose, treat, prevent, and/or amelioratetype II diabetes mellitus (insulin resistant diabetes mellitus).

Additionally, in other embodiments, the polynucleotides and/orpolypeptides corresponding to this gene and/or antagonists thereof(especially neutralizing or antagonistic antibodies) may be used todiagnose, prognose, treat, prevent, and/or ameliorate conditionsassociated with (type I or type II) diabetes mellitus, including, butnot limited to, diabetic ketoacidosis, diabetic coma, nonketotichyperglycemic-hyperosmolar coma, seizures, mental confusion, drowsiness,cardiovascular disease (e.g., heart disease, atherosclerosis,microvascular disease, hypertension, stroke, and other diseases anddisorders as described in the “Cardiovascular Disorders” section),dyslipidemia, kidney disease (e.g., renal failure, nephropathy otherdiseases and disorders as described in the “Renal Disorders” section),nerve damage, neuropathy, vision impairment (e.g., diabetic retinopathyand blindness), ulcers and impaired wound healing, infections (e.g.,infectious diseases and disorders as described in the “InfectiousDiseases” section, especially of the urinary tract and skin), carpaltunnel syndrome and Dupuytren's contracture.

In other embodiments, the polynucleotides and/or polypeptidescorresponding to this gene and/or agonists or antagonists thereof areadministered to an animal, preferably a mammal, and most preferably ahuman, in order to regulate the animal's weight. In specific embodimentsthe polynucleotides and/or polypeptides corresponding to this geneand/or agonists or antagonists thereof are administered to an animal,preferably a mammal, and most preferably a human, in order to controlthe animal's weight by modulating a biochemical pathway involvinginsulin. In still other embodiments the polynucleotides and/orpolypeptides corresponding to this gene and/or agonists or antagoniststhereof are administered to an animal, preferably a mammal, and mostpreferably a human, in order to control the animal's weight bymodulating a biochemical pathway involving insulin-like growth factor.

In addition, endocrine system and/or hormone imbalance disorders and/ordiseases may also include disorders and/or diseases of the testes orovaries, including cancer. Other disorders and/or diseases of the testesor ovaries further include, for example, ovarian cancer, polycysticovary syndrome, Klinefelter's syndrome, vanishing testes syndrome(bilateral anorchia), congenital absence of Leydig's cells,cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillaryhaemangioma of the testis (benign), neoplasias of the testis andneo-testis.

Moreover, endocrine system and/or hormone imbalance disorders and/ordiseases may also include disorders and/or diseases such as, forexample, polyglandular deficiency syndromes, pheochromocytoma,neuroblastoma, multiple Endocrine neoplasia, and disorders and/orcancers of endocrine tissues.

In another embodiment, a polypeptide of the invention, orpolynucleotides, antibodies, agonists, or antagonists corresponding tothat polypeptide, may be used to diagnose, prognose, prevent, and/ortreat endocrine diseases and/or disorders associated with the tissue(s)in which the polypeptide of the invention is expressed, including one,two, three, four, five, or more tissues disclosed in Table 1A, column 8(Tissue Distribution Library Code).

Reproductive System Disorders

The polynucleotides or polypeptides, or agonists or antagonists of theinvention may be used for the diagnosis, treatment, or prevention ofdiseases and/or disorders of the reproductive system. Reproductivesystem disorders that can be treated by the compositions of theinvention, include, but are not limited to, reproductive systeminjuries, infections, neoplastic disorders, congenital defects, anddiseases or disorders which result in infertility, complications withpregnancy, labor, or parturition, and postpartum difficulties.

Reproductive system disorders and/or diseases include diseases and/ordisorders of the testes, including testicular atrophy, testicularfeminization, cryptorchism (unilateral and bilateral), anorchia, ectopictestis, epididymitis and orchitis (typically resulting from infectionssuch as, for example, gonorrhea, mumps, tuberculosis, and syphilis),testicular torsion, vasitis nodosa, germ cell tumors (e.g., seminomas,embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sactumors, and teratomas), stromal tumors (e.g., Leydig cell tumors),hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, anddisorders of sperm production (e.g., immotile cilia syndrome, aspermia,asthenozoospermia, azoospermia, oligospermia, and teratozoospermia).

Reproductive system disorders also include disorders of the prostategland, such as acute non-bacterial prostatitis, chronic non-bacterialprostatitis, acute bacterial prostatitis, chronic bacterial prostatitis,prostatodystonia, prostatosis, granulomatous prostatitis, malacoplakia,benign prostatic hypertrophy or hyperplasia, and prostate neoplasticdisorders, including adenocarcinomas, transitional cell carcinomas,ductal carcinomas, and squamous cell carcinomas.

Additionally, the compositions of the invention may be useful in thediagnosis, treatment, and/or prevention of disorders or diseases of thepenis and urethra, including inflammatory disorders, such asbalanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis,syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis,chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome,condyloma acuminatum, condyloma latum, and pearly penile papules;urethral abnormalities, such as hypospadias, epispadias, and phimosis;premalignant lesions, including Erythroplasia of Queyrat, Bowen'sdisease, Bowenoid paplosis, giant condyloma of Buscke-Lowenstein, andvarrucous carcinoma; penile cancers, including squamous cell carcinomas,carcinoma in situ, verrucous carcinoma, and disseminated penilecarcinoma; urethral neoplastic disorders, including penile urethralcarcinoma, bulbomembranous urethral carcinoma, and prostatic urethralcarcinoma; and erectile disorders, such as priapism, Peyronie's disease,erectile dysfunction, and impotence.

Moreover, diseases and/or disorders of the vas deferens includevasculititis and CBAVD (congenital bilateral absence of the vasdeferens); additionally, the polynucleotides, polypeptides, and agonistsor antagonists of the present invention may be used in the diagnosis,treatment, and/or prevention of diseases and/or disorders of the seminalvesicles, including hydatid disease, congenital chloride diarrhea, andpolycystic kidney disease.

Other disorders and/or diseases of the male reproductive system include,for example, Klinefelter's syndrome, Young's syndrome, prematureejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome,high fever, multiple sclerosis, and gynecomastia.

Further, the polynucleotides, polypeptides, and agonists or antagonistsof the present invention may be used in the diagnosis, treatment, and/orprevention of diseases and/or disorders of the vagina and vulva,including bacterial vaginosis, candida vaginitis, herpes simplex virus,chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, humanpapillomavirus, vaginal trauma, vulvar trauma, adenosis, chlamydiavaginitis, gonorrhea, trichomonas vaginitis, condyloma acuminatum,syphilis, molluscum contagiosum, atrophic vaginitis, Paget's disease,lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome,vaginismus, vulvovaginitis, vulvar vestibulitis, and neoplasticdisorders, such as squamous cell hyperplasia, clear cell carcinoma,basal cell carcinoma, melanomas, cancer of Bartholin's gland, and vulvarintraepithelial neoplasia.

Disorders and/or diseases of the uterus include dysmenorrhea,retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatorybleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman'ssyndrome, premature menopause, precocious puberty, uterine polyps,dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals),and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, andsarcomas. Additionally, the polypeptides, polynucleotides, or agonistsor antagonists of the invention may be useful as a marker or detectorof, as well as in the diagnosis, treatment, and/or prevention ofcongenital uterine abnormalities, such as bicornuate uterus, septateuterus, simple unicornuate uterus, unicornuate uterus with a noncavitaryrudimentary horn, unicornuate uterus with a non-communicating cavitaryrudimentary horn, unicornuate uterus with a communicating cavitary horn,arcuate uterus, uterine didelfus, and T-shaped uterus.

Ovarian diseases and/or disorders include anovulation, polycystic ovarysyndrome (Stein-Leventhal syndrome), ovarian cysts, ovarianhypofunction, ovarian insensitivity to gonadotropins, ovarianoverproduction of androgens, right ovarian vein syndrome, amenorrhea,hirutism, and ovarian cancer (including, but not limited to, primary andsecondary cancerous growth, Sertoli-Leydig tumors, endometriod carcinomaof the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinousadenocarcinoma, and Ovarian Krukenberg tumors).

Cervical diseases and/or disorders include cervicitis, chroniccervicitis, mucopurulent cervicitis, cervical dysplasia, cervicalpolyps, Nabothian cysts, cervical erosion, cervical incompetence, andcervical neoplasms (including, for example, cervical carcinoma, squamousmetaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, andcolumnar cell neoplasia).

Additionally, diseases and/or disorders of the reproductive systeminclude disorders and/or diseases of pregnancy, including miscarriageand stillbirth, such as early abortion, late abortion, spontaneousabortion, induced abortion, therapeutic abortion, threatened abortion,missed abortion, incomplete abortion, complete abortion, habitualabortion, missed abortion, and septic abortion; ectopic pregnancy,anemia, Rh incompatibility, vaginal bleeding during pregnancy,gestational diabetes, intrauterine growth retardation, polyhydramnios,HELLP syndrome, abruptio placentae, placenta previa, hyperemesis,preeclampsia, eclampsia, herpes gestationis, and urticaria of pregnancy.Additionally, the polynucleotides, polypeptides, and agonists orantagonists of the present invention may be used in the diagnosis,treatment, and/or prevention of diseases that can complicate pregnancy,including heart disease, heart failure, rheumatic heart disease,congenital heart disease, mitral valve prolapse, high blood pressure,anemia, kidney disease, infectious disease (e.g., rubella,cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HMV,AIDS, and genital herpes), diabetes mellitus, Graves' disease,thyroiditis, hypothyroidism, Hashimoto's thyroiditis, chronic activehepatitis, cirrhosis of the liver, primary biliary cirrhosis, asthma,systemic lupus eryematosis, rheumatoid arthritis, myasthenia gravis,idiopathic thrombocytopenic purpura, appendicitis, ovarian cysts,gallbladder disorders,and obstruction of the intestine.

Complications associated with labor and parturition include prematurerupture of the membranes, pre-term labor, post-term pregnancy,postmaturity, labor that progresses too slowly, fetal distress (e.g.,abnormal heart rate (fetal or maternal), breathing problems, andabnormal fetal position), shoulder dystocia, prolapsed umbilical cord,amniotic fluid embolism, and aberrant uterine bleeding.

Further, diseases and/or disorders of the postdelivery period, includingendometritis, myometritis, parametritis, peritonitis, pelvicthrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis,saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage,and inverted uterus.

Other disorders and/or diseases of the female reproductive system thatmay be diagnosed, treated, and/or prevented by the polynucleotides,polypeptides, and agonists or antagonists of the present inventioninclude, for example, Turner's syndrome, pseudohermaphroditism,premenstrual syndrome, pelvic inflammatory disease, pelvic congestion(vascular engorgement), frigidity, anorgasmia, dyspareunia, rupturedfallopian tube, and Mittelschmerz.

Infectious Disease

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention can be used to treat or detect infectious agents.For example, by increasing the immune response, particularly increasingthe proliferation and differentiation of B and/or T cells, infectiousdiseases may be treated. The immune response may be increased by eitherenhancing an existing immune response, or by initiating a new immuneresponse. Alternatively, polynucleotides or polypeptides, as well asagonists or antagonists of the present invention may also directlyinhibit the infectious agent, without necessarily eliciting an immuneresponse.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated or detected by a polynucleotide orpolypeptide and/or agonist or antagonist of the present invention.Examples of viruses, include, but are not limited to Examples ofviruses, include, but are not limited to the following DNA and RNAviruses and viral families: Arbovirus, Adenoviridae, Arenaviridae,Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae,Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae(Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex,Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, andparainfluenza), Papiloma virus, Papovaviridae, Parvoviridae,Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae(e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), andTogaviridae (e.g., Rubivirus). Viruses falling within these families cancause a variety of diseases or symptoms, including, but not limited to:arthritis, bronchiollitis, respiratory syncytial virus, encephalitis,eye infections (e.g., conjunctivitis, keratitis), chronic fatiguesyndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese Bencephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever,meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt'sLymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza,Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitteddiseases, skin diseases (e.g., Kaposi's, warts), and viremia.polynucleotides or polypeptides, or agonists or antagonists of theinvention, can be used to treat or detect any of these symptoms ordiseases. In specific embodiments, polynucleotides, polypeptides, oragonists or antagonists of the invention are used to treat: meningitis,Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additionalspecific embodiment polynucleotides, polypeptides, or agonists orantagonists of the invention are used to treat patients nonresponsive toone or more other commercially available hepatitis vaccines. In afurther specific embodiment polynucleotides, polypeptides, or agonistsor antagonists of the invention are used to treat AIDS.

Similarly, bacterial and fungal agents that can cause disease orsymptoms and that can be treated or detected by a polynucleotide orpolypeptide and/or agonist or antagonist of the present inventioninclude, but not limited to, the following Gram-Negative andGram-positive bacteria, bacterial families, and fungi: Actinomyces(e.g., Norcardia), Acinetobacter, Cryptococcus neoformans, Aspergillus,Bacillaceae (e.g., Bacillus anthrasis), Bacteroides (e.g., Bacteroidesfragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borreliaburgdorferi), Brucella, Candidia, Campylobacter, Chlamydia, Clostridium(e.g., Clostridium botulinum, Clostridium dificile, Clostridiumperfringens, Clostridium tetani), Coccidioides, Corynebacterium (e.g.,Corynebacterium diptheriae), Cryptococcus, Dermatocycoses, E. coli(e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacter (e.g. Enterobacter aerogenes), Enterobacteriaceae(Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis,Salmonella typhi), Serratia, Yersinia, Shigella), Erysipelothrix,Haemophilus (e.g., Haemophilus influenza type B), Helicobacter,Legionella (e.g., Legionella pneumophila), Leptospira, Listeria (e.g.,Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacteriumleprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio cholerae),Neisseriaceae (e.g., Neisseria gonorrhea, Neisseria meningitidis),Pasteurellacea, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa),Rickettsiaceae, Spirochetes (e.g., Treponema spp., Leptospira spp.,Borrelia spp.), Shigella spp., Staphylococcus (e.g., Staphylococcusaureus), Meningiococcus, Pneumococcus and Streptococcus (e.g.,Streptococcus pneumoniae and Groups A, B, and C Streptococci), andUreaplasmas. These bacterial, parasitic, and fungal families can causediseases or symptoms, including, but not limited to:antibiotic-resistant infections, bacteremia, endocarditis, septicemia,eye infections (e.g., conjunctivitis), uveitis, tuberculosis,gingivitis, bacterial diarrhea, opportunistic infections (e.g., AIDSrelated infections), paronychia, prosthesis-related infections, dentalcaries, Reiter's Disease, respiratory tract infections, such as WhoopingCough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, dysentery,paratyphoid fever, food poisoning, Legionella disease, chronic and acuteinflammation, erythema, yeast infections, typhoid, pneumonia, gonorrhea,meningitis (e.g., mengitis types A and B), chlamydia, syphillis,diphtheria, leprosy, brucellosis, peptic ulcers, anthrax, spontaneousabortions, birth defects, pneumonia, lung infections, ear infections,deafness, blindness, lethargy, malaise, vomiting, chronic diarrhea,Crohn's disease, colitis, vaginosis, sterility, pelvic inflammatorydiseases, candidiasis, paratuberculosis, tuberculosis, lupus, botulism,gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexuallytransmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses),toxemia, urinary tract infections, wound infections, noscomialinfections. Polynucleotides or polypeptides, agonists or antagonists ofthe invention, can be used to treat or detect any of these symptoms ordiseases. In specific embodiments, polynucleotides, polypeptides,agonists or antagonists of the invention are used to treat: tetanus,diptheria, botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can betreated, prevented, and/or diagnosed by a polynucleotide or polypeptideand/or agonist or antagonist of the present invention include, but notlimited to, the following families or class: Amebiasis, Babesiosis,Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,Giardias, Helminthiasis, Leishmaniasis, Schistisoma, Theileriasis,Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g.,Plasmodium virax, Plasmodium falciparium, Plasmodium malariae andPlasmodium ovale). These parasites can cause a variety of diseases orsymptoms, including, but not limited to: Scabies, Trombiculiasis, eyeinfections, intestinal disease (e.g., dysentery, giardiasis), liverdisease, lung disease, opportunistic infections (e.g., AIDS related),malaria, pregnancy complications, and toxoplasmosis. polynucleotides orpolypeptides, or agonists or antagonists of the invention, can be usedto treat, prevent, and/or diagnose any of these symptoms or diseases. Inspecific embodiments, polynucleotides, polypeptides, or agonists orantagonists of the invention are used to treat, prevent, and/or diagnosemalaria.

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention of the present invention could either be byadministering an effective amount of a polypeptide to the patient, or byremoving cells from the patient, supplying the cells with apolynucleotide of the present invention, and returning the engineeredcells to the patient (ex vivo therapy). Moreover, the polypeptide orpolynucleotide of the present invention can be used as an antigen in avaccine to raise an immune response against infectious disease.

Regeneration

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention can be used to differentiate, proliferate, andattract cells, leading to the regeneration of tissues. (See, Science276:59-87 (1997)). The regeneration of tissues could be used to repair,replace, or protect tissue damaged by congenital defects, trauma(wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis,osteocarthritis, periodontal disease, liver failure), surgery, includingcosmetic plastic surgery, fibrosis, reperfusion injury, or systemiccytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac), vasculature (including vascularand lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage,tendon, and ligament) tissue. Preferably, regeneration occurs without ordecreased scarring. Regeneration also may include angiogenesis.

Moreover, polynucleotides or polypeptides, as well as agonists orantagonists of the present invention, may increase regeneration oftissues difficult to heal. For example, increased tendon/ligamentregeneration would quicken recovery time after damage. Polynucleotidesor polypeptides, as well as agonists or antagonists of the presentinvention could also be used prophylactically in an effort to avoiddamage. Specific diseases that could be treated include of tendinitis,carpal tunnel syndrome, and other tendon or ligament defects. A furtherexample of tissue regeneration of non-healing wounds includes pressureulcers, ulcers associated with vascular insufficiency, surgical, andtraumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by usingpolynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention, to proliferate and differentiate nerve cells.Diseases that could be treated using this method include central andperipheral nervous system diseases, neuropathies, or mechanical andtraumatic disorders (e.g., spinal cord disorders, head trauma,cerebrovascular disease, and stoke). Specifically, diseases associatedwith peripheral nerve injuries, peripheral neuropathy (e.g., resultingfrom chemotherapy or other medical therapies), localized neuropathies,and central nervous system diseases (e.g., Alzheimer's disease,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome), could all be treated using thepolynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention.

Gastrointestinal Disorders

Polynucleotides or polypeptides, or agonists or antagonists of thepresent invention, may be used to treat, prevent, diagnose, and/orprognose gastrointestinal disorders, including inflammatory diseasesand/or conditions, infections, cancers (e.g., intestinal neoplasms(carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of thesmall intestine, small bowl lymphoma)), and ulcers, such as pepticulcers.

Gastrointestinal disorders include dysphagia, odynophagia, inflammationof the esophagus, peptic esophagitis, gastric reflux, submucosalfibrosis and stricturing, Mallory-Weiss lesions, leiomyomas, lipomas,epidermal cancers, adeoncarcinomas, gastric retention disorders,gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps of thestomach, autoimmune disorders such as pernicious anemia, pyloricstenosis, gastritis (bacterial, viral, eosinophilic, stress-induced,chronic erosive, atrophic, plasma cell, and Ménétrier's), and peritonealdiseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst,mesenteric lymphadenitis, mesenteric vascular occlusion, panniculitis,neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess,).

Gastrointestinal disorders also include disorders associated with thesmall intestine, such as malabsorption syndromes, distension, irritablebowel syndrome, sugar intolerance, celiac disease, duodenal ulcers,duodenitis, tropical sprue, Whipple's disease, intestinallymphangiectasia, Crohn's disease, appendicitis, obstructions of theileum, Meckel's diverticulum, multiple diverticula, failure of completerotation of the small and large intestine, lymphoma, and bacterial andparasitic diseases (such as Traveler's diarrhea, typhoid andparatyphoid, cholera, infection by Roundworms (Ascariasis lumbricoides),Hookworms (Ancylostoma duodenale), Threadworms (Enterobiusvermicularis), Tapeworms (Taenia saginata, Echinococcus granulosus,Diphyllobothrium spp., and T. solium).

Liver diseases and/or disorders include intrahepatic cholestasis(alagille syndrome, biliary liver cirrhosis), fatty liver (alcoholicfatty liver, reye syndrome), hepatic vein thrombosis, hepatolentriculardegeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenalsyndrome, portal hypertension (esophageal and gastric varices), liverabscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary andexperimental), alcoholic liver diseases (fatty liver, hepatitis,cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebicliver abscess), jaundice (hemolytic, hepatocellular, and cholestatic),cholestasis, portal hypertension, liver enlargement, ascites, hepatitis(alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune,hepatitis B, hepatitis C, hepatitis D, drug induced), toxic hepatitis,viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitisD, hepatitis E), Wilson's disease, granulomatous hepatitis, secondarybiliary cirrhosis, hepatic encephalopathy, portal hypertension, varices,hepatic encephalopathy, primary biliary cirrhosis, primary sclerosingcholangitis, hepatocellular adenoma, hemangiomas, bile stones, liverfailure (hepatic encephalopathy, acute liver failure), and liverneoplasms (angiomyolipoma, calcified liver metastases, cystic livermetastases, epithelial tumors, fibrolamellar hepatocarcinoma, focalnodular hyperplasia, hepatic adenoma, hepatobiliary cystadenoma,hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, liverhemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors ofliver, nodular regenerative hyperplasia, benign liver tumors (Hepaticcysts [Simple cysts, Polycystic liver disease, Hepatobiliarycystadenoma, Choledochal cyst], Mesenchymal tumors [Mesenchymalhamartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis,Lipomas, Inflammatory pseudotumor, Miscellaneous], Epithelial tumors[Bile duct epithelium (Bile duct hamartoma, Bile duct adenoma),Hepatocyte (Adenoma, Focal nodular hyperplasia, Nodular regenerativehyperplasia)], malignant liver tumors [hepatocellular, hepatoblastoma,hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma,cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi'ssarcoma, hemangioendothelioma, other tumors, embryonal sarcoma,fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma,teratoma, carcinoid, squamous carcinoma, primary lymphoma]), peliosishepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittentporphyria, porphyria cutanea tarda), Zellweger syndrome).

Pancreatic diseases and/or disorders include acute pancreatitis, chronicpancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis),neoplasms (adenocarcinoma of the pancreas, cystadenocarcinoma,insulinoma, gastrinoma, and glucagonoma, cystic neoplasms, islet-celltumors, pancreoblastoma), and other pancreatic diseases (e.g., cysticfibrosis, cyst (pancreatic pseudocyst, pancreatic fistula,insufficiency)).

Gallbladder diseases include gallstones (cholelithiasis andcholedocholithiasis), postcholecystectomy syndrome, diverticulosis ofthe gallbladder, acute cholecystitis, chronic cholecystitis, bile ducttumors, and mucocele.

Diseases and/or disorders of the large intestine includeantibiotic-associated colitis, diverticulitis, ulcerative colitis,acquired megacolon, abscesses, fungal and bacterial infections,anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases(colitis, colonic neoplasms [colon cancer, adenomatous colon polyps(e.g., villous adenoma), colon carcinoma, colorectal cancer], colonicdiverticulitis, colonic diverticulosis, megacolon [Hirschsprung disease,toxic megacolon]; sigmoid diseases [proctocolitis, sigmoin neoplasms]),constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery),duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenalulcer, duodenitis), enteritis (enterocolitis), HIV enteropathy, ilealdiseases (ileal neoplasms, ileitis), immunoproliferative smallintestinal disease, inflammatory bowel disease (ulcerative colitis,Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis,balantidiasis, blastocystis infections, cryptosporidiosis,dientamoebiasis, amebic dysentery, giardiasis), intestinal fistula(rectal fistula), intestinal neoplasms (cecal neoplasms, colonicneoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps,jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferentloop syndrome, duodenal obstruction, impacted feces, intestinalpseudo-obstruction [cecal volvulus], intussusception), intestinalperforation, intestinal polyps (colonic polyps, gardner syndrome,peutz-jeghers syndrome), jejunal diseases (jejunal neoplasms),malabsorption syndromes (blind loop syndrome, celiac disease, lactoseintolerance, short bowl syndrome, tropical sprue, whipple's disease),mesenteric vascular occlusion, pneumatosis cystoides intestinalis,protein-losing enteropathies (intestinal lymphagiectasis), rectaldiseases (anus diseases, fecal incontinence, hemorrhoids, proctitis,rectal fistula, rectal prolapse, rectocele), peptic ulcer (duodenalulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer,Zollinger-Ellison syndrome), postgastrectomy syndromes (dumpingsyndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux(bile reflux), gastric antral vascular ectasia, gastric fistula, gastricoutlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis,stomach dilatation, stomach diverticulum, stomach neoplasms (gastriccancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastricpolyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis,visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum,postoperative nausea and vomiting) and hemorrhagic colitis.

Further diseases and/or disorders of the gastrointestinal system includebiliary tract diseases, such as, gastroschisis, fistula (e.g., biliaryfistula, esophageal fistula, gastric fistula, intestinal fistula,pancreatic fistula), neoplasms (e.g., biliary tract neoplasms,esophageal neoplasms, such as adenocarcinoma of the esophagus,esophageal squamous cell carcinoma, gastrointestinal neoplasms,pancreatic neoplasms, such as adenocarcinoma of the pancreas, mucinouscystic neoplasm of the pancreas, pancreatic cystic neoplasms,pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g.,bullous diseases, candidiasis, glycogenic acanthosis, ulceration,barrett esophagus varices, atresia, cyst, diverticulum (e.g., Zenker'sdiverticulum), fistula (e.g., tracheoesophageal fistula), motilitydisorders (e.g., CREST syndrome, deglutition disorders, achalasia,spasm, gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaavesyndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatichernia (e.g., hiatal hernia); gastrointestinal diseases, such as,gastroenteritis (e.g., cholera morbus, norwalk virus infection),hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomachneoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma,stomach cancer)), hernia (e.g., congenital diaphragmatic hernia, femoralhernia, inguinal hernia, obturator hernia, umbilical hernia, ventralhernia), and intestinal diseases (e.g., cecal diseases (appendicitis,cecal neoplasms)).

Chemotaxis

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention may have chemotaxis activity. A chemotaxicmolecule attracts or mobilizes cells (e.g., monocytes, fibroblasts,neutrophils, T-cells, mast cells, eosinophils, epithelial and/orendothelial cells) to a particular site in the body, such asinflammation, infection, or site of hyperproliferation. The mobilizedcells can then fight off and/or heal the particular trauma orabnormality.

Polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention may increase chemotaxic activity of particularcells. These chemotactic molecules can then be used to treatinflammation, infection, hyperproliferative disorders, or any immunesystem disorder by increasing the number of cells targeted to aparticular location in the body. For example, chemotaxic molecules canbe used to treat wounds and other trauma to tissues by attracting immunecells to the injured location. Chemotactic molecules of the presentinvention can also attract fibroblasts, which can be used to treatwounds.

It is also contemplated that polynucleotides or polypeptides, as well asagonists or antagonists of the present invention may inhibit chemotacticactivity. These molecules could also be used to treat disorders. Thus,polynucleotides or polypeptides, as well as agonists or antagonists ofthe present invention could be used as an inhibitor of chemotaxis.

Binding Activity

A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors), orsmall molecules.

Preferably, the molecule is closely related to the natural ligand of thepolypeptide, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2):Chapter 5 (1991)). Similarly, themolecule can be closely related to the natural receptor to which thepolypeptide binds, or at least, a fragment of the receptor capable ofbeing bound by the polypeptide (e.g., active site). In either case, themolecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide. Preferred cells includecells from mammals, yeast, Drosophila, or E. coli. Cells expressing thepolypeptide (or cell membrane containing the expressed polypeptide) arethen preferably contacted with a test compound potentially containingthe molecule to observe binding, stimulation, or inhibition of activityof either the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

Preferably, an ELISA assay can measure polypeptide level or activity ina sample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure polypeptide level or activity byeither binding, directly or indirectly, to the polypeptide or bycompeting with the polypeptide for a substrate.

Additionally, the receptor to which the polypeptide of the presentinvention binds can be identified by numerous methods known to those ofskill in the art, for example, ligand panning and FACS sorting (Coligan,et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Forexample, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the polypeptides, for example, NIH3T3cells which are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labeled. The polypeptides can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of thepolypeptide of the present invention thereby effectively generatingagonists and antagonists of the polypeptide of the present invention.See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721,5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82(1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); andLorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998); each ofthese patents and publications are hereby incorporated by reference). Inone embodiment, alteration of polynucleotides and correspondingpolypeptides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments into a desired molecule byhomologous, or site-specific, recombination. In another embodiment,polynucleotides and corresponding polypeptides may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of the polypeptide of the present invention may berecombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules. Inpreferred embodiments, the heterologous molecules are family members. Infurther preferred embodiments, the heterologous molecule is a growthfactor such as, for example, platelet-derived growth factor (PDGF),insulin-like growth factor (IGF-I), transforming growth factor(TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor(FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5,BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2,dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of thepolypeptide of the present invention. Biologically active fragments arethose exhibiting activity similar, but not necessarily identical, to anactivity of the polypeptide of the present invention. The biologicalactivity of the fragments may include an improved desired activity, or adecreased undesirable activity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, a the polypeptide of the present invention,the compound to be screened and ³[H] thymidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of ³[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of ³[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the receptor is measured and the ability of the compound tobind to the receptor and elicit a second messenger response is measuredto determine if the compound is a potential agonist or antagonist. Suchsecond messenger systems include but are not limited to, cAMP guanylatecyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting thepolypeptide/molecule. Moreover, the assays can discover agents which mayinhibit or enhance the production of the polypeptides of the inventionfrom suitably manipulated cells or tissues.

Therefore, the invention includes a method of identifying compoundswhich bind to a polypeptide of the invention comprising the steps of:(a) incubating a candidate binding compound with a polypeptide of thepresent invention; and (b) determining if binding has occurred.Moreover, the invention includes a method of identifyingagonists/antagonists comprising the steps of: (a) incubating a candidatecompound with a polypeptide of the present invention, (b) assaying abiological activity, and (b) determining if a biological activity of thepolypeptide has been altered.

Targeted Delivery

In another embodiment, the invention provides a method of deliveringcompositions to targeted cells expressing a receptor for a polypeptideof the invention, or cells expressing a cell bound form of a polypeptideof the invention.

As discussed herein, polypeptides or antibodies of the invention may beassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions. In one embodiment, the invention provides a method for thespecific delivery of compositions of the invention to cells byadministering polypeptides of the invention (including antibodies) thatare associated with heterologous polypeptides or nucleic acids. In oneexample, the invention provides a method for delivering a therapeuticprotein into the targeted cell. In another example, the inventionprovides a method for delivering a single stranded nucleic acid (e.g.,antisense or ribozymes) or double stranded nucleic acid (e.g., DNA thatcan integrate into the cell's genome or replicate episomally and thatcan be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the presentinvention, or the polynucleotides encoding these polypeptides, to screenfor molecules which modify the activities of the polypeptides of thepresent invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs orany other agents which affect activities mediated by the polypeptides ofthe present invention. These methods comprise contacting such an agentwith a polypeptide of the present invention or a fragment thereof andassaying for the presence of a complex between the agent and thepolypeptide or a fragment thereof, by methods well known in the art. Insuch a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the polypeptides ofthe present invention, and is described in great detail in EuropeanPatent Application 84/03564, published on Sep. 13, 1984, which isincorporated herein by reference herein. Briefly stated, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The peptide testcompounds are reacted with polypeptides of the present invention andwashed. Bound polypeptides are then detected by methods well known inthe art. Purified polypeptides are coated directly onto plates for usein the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding polypeptidesof the present invention specifically compete with a test compound forbinding to the polypeptides or fragments thereof. In this manner, theantibodies are used to detect the presence of any peptide which sharesone or more antigenic epitopes with a polypeptide of the invention.

Polypeptides of the Invention Binding Peptides and Other Molecules

The invention also encompasses screening methods for identifyingpolypeptides and nonpolypeptides that bind polypeptides of theinvention, and the polypeptide of the invention binding moleculesidentified thereby. These binding molecules are useful, for example, asagonists and antagonists of the polypeptides of the invention. Suchagonists and antagonists can be used, in accordance with the invention,in the therapeutic embodiments described in detail, below.

This method comprises the steps of:contacting a polypeptide of theinvention with a plurality of molecules; and identifying a molecule thatbinds the polypeptide of the invention.

The step of contacting the polypeptide of the invention with theplurality of molecules may be effected in a number of ways. For example,one may contemplate immobilizing the polypeptide of the invention on asolid support and bringing a solution of the plurality of molecules incontact with the immobilized polypeptide of the invention. Such aprocedure would be akin to an affinity chromatographic process, with theaffinity matrix being comprised of the immobilized polypeptide of theinvention. The molecules having a selective affinity for the polypeptideof the invention can then be purified by affinity selection. The natureof the solid support, process for attachment of the polypeptide of theinvention to the solid support, solvent, and conditions of the affinityisolation or selection are largely conventional and well known to thoseof ordinary skill in the art.

Alternatively, one may also separate a plurality of polypeptides intosubstantially separate fractions comprising a subset of or individualpolypeptides. For instance, one can separate the plurality ofpolypeptides by gel electrophoresis, column chromatography, or likemethod known to those of ordinary skill for the separation ofpolypeptides. The individual polypeptides can also be produced by atransformed host cell in such a way as to be expressed on or about itsouter surface (e.g., a recombinant phage). Individual isolates can thenbe “probed” by the polypeptide of the invention, optionally in thepresence of an inducer should one be required for expression, todetermine if any selective affinity interaction takes place between thepolypeptide of the invention and the individual clone. Prior tocontacting the polypeptide of the invention with each fractioncomprising individual polypeptides, the polypeptides could first betransferred to a solid support for additional convenience. Such a solidsupport may simply be a piece of filter membrane, such as one made ofnitrocellulose or nylon. In this manner, positive clones could beidentified from a collection of transformed host cells of an expressionlibrary, which harbor a DNA construct encoding a polypeptide having aselective affinity for a polypeptide of the invention. Furthermore, theamino acid sequence of the polypeptide having a selective affinity forthe polypeptide of the invention can be determined directly byconventional means or the coding sequence of the DNA encoding thepolypeptide can frequently be determined more conveniently. The primarysequence can then be deduced from the corresponding DNA sequence. If theamino acid sequence is to be determined from the polypeptide itself, onemay use microsequencing techniques. The sequencing technique may includemass spectroscopy.

In certain situations, it may be desirable to wash away any unboundpolypeptide of the invention, or alterntatively, unbound polypeptides,from a mixture of the polypeptide of the invention and the plurality ofpolypeptides prior to attempting to determine or to detect the presenceof a selective affinity interaction. Such a wash step may beparticularly desirable when the polypeptide of the invention or theplurality of polypeptides is bound to a solid support.

The plurality of molecules provided according to this method may beprovided by way of diversity libraries, such as random or combinatorialpeptide or nonpeptide libraries which can be screened for molecules thatspecifically bind to a polypeptide of the invention. Many libraries areknown in the art that can be used, e.g., chemically synthesizedlibraries, recombinant (e.g., phage display libraries), and in vitrotranslation-based libraries. Examples of chemically synthesizedlibraries are described in Fodor et al., 1991, Science 251:767-773;Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature354:82-84; Medynski, 1994, Bio/Technology 12:709-710;Gallop et al.,1994, J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993,Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl.Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner,1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.

Examples of phage display libraries are described in Scott and Smith,1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406;Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra,1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65;and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

In vitro translation-based libraries include but are not limited tothose described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991;and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.

By way of examples of nonpeptide libraries, a benzodiazepine library(see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712)can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc.Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example ofa library that can be used, in which the amide functionalities inpeptides have been permethylated to generate a chemically transformedcombinatorial library, is described by Ostresh et al. (1994, Proc. Natl.Acad. Sci. USA 91:11138-11142).

The variety of non-peptide libraries that are useful in the presentinvention is great. For example, Ecker and Crooke, 1995, Bio/Technology13:351-360 list benzodiazepines, hydantoins, piperazinediones,biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids,acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, andoxazolones as among the chemical species that form the basis of variouslibraries.

Non-peptide libraries can be classified broadly into two types:decorated monomers and oligomers. Decorated monomer libraries employ arelatively simple scaffold structure upon which a variety functionalgroups is added. Often the scaffold will be a molecule with a knownuseful pharmacological activity. For example, the scaffold might be thebenzodiazepine structure.

Non-peptide oligomer libraries utilize a large number of monomers thatare assembled together in ways that create new shapes that depend on theorder of the monomers. Among the monomer units that have been used arecarbamates, pyrrolinones, and morpholinos. Peptoids, peptide-likeoligomers in which the side chain is attached to the alpha amino grouprather than the alpha carbon, form the basis of another version ofnon-peptide oligomer libraries. The first non-peptide oligomer librariesutilized a single type of monomer and thus contained a repeatingbackbone. Recent libraries have utilized more than one monomer, givingthe libraries added flexibility.

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, 1989, Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390;Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992,Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992,Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. Nos.5,096,815, 5,223,409, and 5,198,346, all to Ladner et al.; Rebar andPabo, 1993, Science 263:671-673; and CT Publication No. WO 94/18318.

In a specific embodiment, screening to identify a molecule that binds apolypeptide of the invention can be carried out by contacting thelibrary members with a polypeptide of the invention immobilized on asolid phase and harvesting those library members that bind to thepolypeptide of the invention. Examples of such screening methods, termed“panning” techniques are described by way of example in Parmley andSmith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques13:422-427; PCT Publication No. WO 94/18318; and in references citedherein.

In another embodiment, the two-hybrid system for selecting interactingproteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien etal., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used toidentify molecules that specifically bind to a polypeptide of theinvention.

Where the polypeptide of the invention binding molecule is apolypeptide, the polypeptide can be conveniently selected from anypeptide library, including random peptide libraries, combinatorialpeptide libraries, or biased peptide libraries. The term “biased” isused herein to mean that the method of generating the library ismanipulated so as to restrict one or more parameters that govern thediversity of the resulting collection of molecules, in this casepeptides.

Thus, a truly random peptide library would generate a collection ofpeptides in which the probability of finding a particular amino acid ata given position of the peptide is the same for all 20 amino acids. Abias can be introduced into the library, however, by specifying, forexample, that a lysine occur every fifth amino acid or that positions 4,8, and 9 of a decapeptide library be fixed to include only arginine.Clearly, many types of biases can be contemplated, and the presentinvention is not restricted to any particular bias. Furthermore, thepresent invention contemplates specific types of peptide libraries, suchas phage displayed peptide libraries and those that utilize a DNAconstruct comprising a lambda phage vector with a DNA insert.

As mentioned above, in the case of a polypeptide of the inventionbinding molecule that is a polypeptide, the polypeptide may have about 6to less than about 60 amino acid residues, preferably about 6 to about10 amino acid residues, and most preferably, about 6 to about 22 aminoacids. In another embodiment, a polypeptide of the invention bindingpolypeptide has in the range of 15-100 amino acids, or 20-50 aminoacids.

The selected polypeptide of the invention binding polypeptide can beobtained by chemical synthesis or recombinant expression.

Antisense and Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in SEQ IDNO:X, or the complementary strand thereof, and/or to nucleotidesequences contained a deposited clone. In one embodiment, antisensesequence is generated internally by the organism, in another embodiment,the antisense sequence is separately administered (see, for example,O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as AnitsenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Antisense technology can be used to control gene expression throughantisense DNA or RNA, or through triple-helix formation. Antisensetechniques are discussed for example, in Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research,6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan etal., Science, 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs toinhibit the growth of the non-lymphocytic leukemia cell line HL-60 andother cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcoR1 site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5,10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligatedto the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the antisense nucleic acid of the invention.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding a polypeptide of the invention, orfragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinsteret al., Nature, 296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a gene ofinterest. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded antisense nucleic acids of the invention, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acidGenerally, the larger the hybridizing nucleic acid, the more basemismatches with a RNA sequence of the invention it may contain and stillform a stable duplex (or triplex as the case may be). One skilled in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature,372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′-non-translated, non-coding regions of a polynucleotidesequence of the invention could be used in an antisense approach toinhibit translation of endogenous mRNA. Oligonucleotides complementaryto the 5′ untranslated region of the mRNA should include the complementof the AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the invention. Whether designed to hybridize tothe 5′-, 3′- or coding region of mRNA, antisense nucleic acids should beat least six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A.86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652(1987); PCT Publication NO: WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication NO: WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or intercalatingagents. (See, e.g., Zon, Pharm. Res., 5:539-549 (1988)). To this end,the oligonucleotide may be conjugated to another molecule, e.g., apeptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res., 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci.U.S.A., 85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the coding region sequenceof the invention could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science, 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy mRNAs corresponding to thepolynucleotides of the invention, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within eachnucleotide sequence disclosed in the sequence listing. Preferably, theribozyme is engineered so that the cleavage recognition site is locatednear the 5′ end of the mRNA corresponding to the polynucleotides of theinvention; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express thepolynucleotides of the invention in vivo. DNA constructs encoding theribozyme may be introduced into the cell in the same manner as describedabove for the introduction of antisense encoding DNA. A preferred methodof delivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive promoter, such as, for example, polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous messages andinhibit translation. Since ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth ofscar tissue during wound healing.

The antagonist/agonist may also be employed to treat, prevent, and/ordiagnose the diseases described herein.

Thus, the invention provides a method of treating or preventingdiseases, disorders, and/or conditions, including but not limited to thediseases, disorders, and/or conditions listed throughout thisapplication, associated with overexpression of a polynucleotide of thepresent invention by administering to a patient (a) an antisensemolecule directed to the polynucleotide of the present invention, and/or(b) a ribozyme directed to the polynucleotide of the present invention.

invention, and/or (b) a ribozyme directed to the polynucleotide of thepresent invention

Other Activities

The polypeptide of the present invention, as a result of the ability tostimulate vascular endothelial cell growth, may be employed in treatmentfor stimulating re-vascularization of ischemic tissues due to variousdisease conditions such as thrombosis, arteriosclerosis, and othercardiovascular conditions. These polypeptide may also be employed tostimulate angiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulateneuronal growth and to treat, prevent, and/or diagnose neuronal damagewhich occurs in certain neuronal disorders or neuro-degenerativeconditions such as Alzheimer's disease, Parkinson's disease, andAIDS-related complex. The polypeptide of the invention may have theability to stimulate chondrocyte growth, therefore, they may be employedto enhance bone and periodontal regeneration and aid in tissuetransplants or bone grafts.

The polypeptide of the present invention may be also be employed toprevent skin aging due to sunburn by stimulating keratinocyte growth.

The polypeptide of the invention may also be employed for preventinghair loss, since FGF family members activate hair-forming cells andpromotes melanocyte growth. Along the same lines, the polypeptides ofthe present invention may be employed to stimulate growth anddifferentiation of hematopoietic cells and bone marrow cells when usedin combination with other cytokines.

The polypeptide of the invention may also be employed to maintain organsbefore transplantation or for supporting cell culture of primarytissues.

The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

The polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

The polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to modulate mammaliancharacteristics, such as body height, weight, hair color, eye color,skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,cosmetic surgery). Similarly, polypeptides or polynucleotides and/oragonist or antagonists of the present invention may be used to modulatemammalian metabolism affecting catabolism, anabolism, processing,utilization, and storage of energy.

A polypeptide, polynucleotide, agonist, or antagonist of the presentinvention may be used to treat weight disorders, including but notlimited to, obesity, cachexia, wasting disease, anorexia, and bulimia.

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may be used to change a mammal's mental state orphysical state by influencing biorhythms, caricadic rhythms, depression(including depressive diseases, disorders, and/or conditions), tendencyfor violence, tolerance for pain, reproductive capabilities (preferablyby Activin or Inhibin-like activity), hormonal or endocrine levels,appetite, libido, memory, stress, or other cognitive qualities.Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used as a food additive or preservative,such as to increase or decrease storage capabilities, fat content,lipid, protein, carbohydrate, vitamins, minerals, cofactors or othernutritional components.

Other Preferred Embodiments

Other preferred embodiments of the claimed invention include an isolatednucleic acid molecule comprising a nucleotide sequence which is at least95% identical to a sequence of at least about 50 contiguous nucleotidesin the nucleotide sequence of SEQ ID NO:X wherein X is any integer asdefined in Table 1A. Also preferred is the above nucleic acid moleculewherein said sequence of contiguous nucleotides is included in thenucleotide sequence of SEQ ID NO:X in the range of positions beginningwith the nucleotide at about the position of the 5′ Nucleotide of theClone Sequence and ending with the nucleotide at about the position ofthe 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X inTable 1A. Further preferred is the above nucleic acid molecule whereinsaid sequence of contiguous nucleotides is included in the nucleotidesequence of SEQ ID NO:X in the range of positions beginning with thenucleotide at about the position of the 5′ Nucleotide of the Start Codonand ending with the nucleotide at about the position of the 3′Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1A.Similarly preferred is the above nucleic acid molecule wherein saidsequence of contiguous nucleotides is included in the nucleotidesequence of SEQ ID NO:X in the range of positions beginning with thenucleotide at about the position of the 5′ Nucleotide of the First AminoAcid of the Signal Peptide and ending with the nucleotide at about theposition of the 3′ Nucleotide of the Clone Sequence as defined for SEQID NO:X in Table 1A.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 150 contiguous nucleotides in the nucleotide sequence of SEQID NO:X.

Further preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 500 contiguous nucleotides in the nucleotide sequence of SEQID NO:X.

A further preferred embodiment is a nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to the nucleotidesequence of SEQ ID NO:X beginning with the nucleotide at about theposition of the 5′ Nucleotide of the First Amino Acid of the SignalPeptide and ending with the nucleotide at about the position of the 3′Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1A.

A further preferred embodiment is an isolated nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thecomplete nucleotide sequence of SEQ ID NO:X.

Also preferred is an isolated nucleic acid molecule which hybridizesunder stringent hybridization conditions to a nucleic acid molecule,wherein said isolated nucleic acid molecule does not hybridize understringent hybridization conditions to a nucleic acid molecule having anucleotide sequence consisting of only A residues or of only T residues.

Also preferred is a composition of matter comprising a DNA moleculewhich comprises a human cDNA clone identified by a cDNA Clone Identifierin Table 1A, which DNA molecule is contained in the material depositedwith the American Type Culture Collection and given the ATCC DepositNumber shown in Table 1A for said cDNA Clone Identifier.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in the nucleotide sequence of the cDNAof a human cDNA clone identified by a cDNA Clone Identifier in Table 1A,which DNA molecule is contained in the deposit given the ATCC DepositNumber shown in Table 1A. Further preferred is the above nucleic acidmolecule, wherein said sequence of at least 50 contiguous nucleotides isincluded in the nucleotide sequence of the complete open reading framesequence encoded by said human cDNA clone. In addition, an isolatednucleic acid molecule of the invention may comprise a nucleotidesequence which is at least 95% identical to sequence of at least 150contiguous nucleotides in the nucleotide sequence of the cDNA in saidhuman cDNA clone. A further preferred embodiment is an isolated nucleicacid molecule comprising a nucleotide sequence which is at least 95%identical to sequence of at least 500 contiguous nucleotides in thenucleotide sequence of the cDNA in said human cDNA clone. A furtherpreferred embodiment is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to the completenucleotide sequence of the cDNA in said human cDNA clone.

A further preferred embodiment is a method for detecting in a biologicalsample a nucleic acid molecule comprising a nucleotide sequence which isat least 95% identical to a sequence of at least 50 contiguousnucleotides in a sequence selected from the group consisting of: anucleotide sequence of SEQ ID NO:X wherein X is any integer as definedin Table 1A; and a nucleotide sequence encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table 1A and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in Table1A; which method comprises: (a) a step of comparing a nucleotidesequence of at least one nucleic acid molecule in said sample with asequence selected from said group; and (b) determining whether thesequence of said nucleic acid molecule in said sample is at least 95%identical to said selected sequence. The step of comparing sequences inthe above method may further comprise determining the extent of nucleicacid hybridization between nucleic acid molecules in said sample and anucleic acid molecule comprising said sequence selected from said group.Similarly, the step of comparing sequences in the above method may beperformed by comparing the nucleotide sequence determined from a nucleicacid molecule in said sample with said sequence selected from saidgroup. The nucleic acid molecules can comprise DNA molecules or RNAmolecules.

A further preferred embodiment is a method for identifying the species,tissue or cell type of a biological sample which method comprises a stepof detecting nucleic acid molecules in said sample, if any, comprising anucleotide sequence that is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from the groupconsisting of: a nucleotide sequence of SEQ ID NO:X wherein X is anyinteger as defined in Table 1A; and a nucleotide sequence encoded by ahuman cDNA clone identified by a cDNA Clone Identifier in Table 1A andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table 1A. This method described above may further comprisea step of detecting nucleic acid molecules comprising a nucleotidesequence in a panel of at least two nucleotide sequences, wherein atleast one sequence in said panel is at least 95% identical to a sequenceof at least 50 contiguous nucleotides in a sequence selected from saidgroup.

Also preferred is a method for diagnosing in a subject a pathologicalcondition associated with abnormal structure or expression of a geneencoding a secreted protein identified in Table 1A, which methodcomprises a step of detecting in a biological sample obtained from saidsubject nucleic acid molecules, if any, comprising a nucleotide sequencethat is at least 95% identical to a sequence of at least 50 contiguousnucleotides in a sequence selected from the group consisting of: anucleotide sequence of SEQ ID NO:X wherein X is any integer as definedin Table 1A; and a nucleotide sequence encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table 1A and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in Table1A. This method described above may further comprise a step of detectingnucleic acid molecules comprising a nucleotide sequence in a panel of atleast two nucleotide sequences, wherein at least one sequence in saidpanel is at least 95% identical to a sequence of at least 50 contiguousnucleotides in a sequence selected from said group.

Also preferred is a composition of matter comprising isolated nucleicacid molecules wherein the nucleotide sequences of said nucleic acidmolecules comprise a panel of at least two nucleotide sequences, whereinat least one sequence in said panel is at least 95% identical to asequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:Xwherein X is any integer as defined in Table 1A; and a nucleotidesequence encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1A and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1A. The nucleic acidmolecules can comprise DNA molecules or RNA molecules.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the amino acid sequence of SEQ ID NO:Y whereinY is any integer as defined in Table 1A. Further preferred is the aboveisolated polypeptide, wherein said sequence of contiguous amino acids isincluded in the amino acid sequence of SEQ ID NO:Y in the range ofpositions beginning with the residue at about the position of the FirstAmino Acid of the Secreted Portion and ending with the residue at aboutthe Last Amino Acid of the Open Reading Frame as set forth for SEQ IDNO:Y in Table 1A.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to the complete amino acid sequence ofSEQ ID NO:Y.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the complete amino acid sequence of a secretedprotein encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1A and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1A. Further preferredis the above isolated polypeptide wherein said sequence of contiguousa-amino acids is included in the amino acid sequence of a secretedportion of the secreted protein encoded by a human cDNA clone identifiedby a cDNA Clone Identifier in Table 1A and contained in the deposit withthe ATCC Deposit Number shown for said cDNA clone in Table 1A.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of the secretedportion of the protein encoded by a human cDNA clone identified by acDNA Clone Identifier in Table 1A and contained in the deposit with theATCC Deposit Number shown for said cDNA clone in Table 1A.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of the secretedportion of the protein encoded by a human cDNA clone identified by acDNA Clone Identifier in Table 1A and contained in the deposit with theATCC Deposit Number shown for said cDNA clone in Table 1A.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to the amino acid sequence of thesecreted portion of the protein encoded by a human cDNA clone identifiedby a cDNA Clone Identifier in Table 1A and contained in the deposit withthe ATCC Deposit Number shown for said cDNA clone in Table 1A.

Further preferred is an isolated antibody which binds specifically to apolypeptide comprising an amino acid sequence that is at least 90%identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:Y wherein Y is any integer as defined in Table 1A; and acomplete amino acid sequence of a protein encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table 1A and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in Table1A.

Further preferred is a method for detecting in a biological sample apolypeptide comprising an amino acid sequence which is at least 90%identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:Y wherein Y is any integer as defined in Table 1A; and acomplete amino acid sequence of a protein encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table 1A and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in Table1A; which method comprises: (a) a step of comparing an amino acidsequence of at least one polypeptide molecule in said sample with asequence selected from said group; and (b) determining whether thesequence of said polypeptide molecule in said sample is at least 90%identical to said sequence of at least 10 contiguous amino acids. Thestep in the above method of comparing an amino acid sequence of at leastone polypeptide molecule in said sample with a sequence selected fromsaid group may further comprise determining the extent of specificbinding of polypeptides in said sample to an antibody which bindsspecifically to a polypeptide comprising an amino acid sequence that isat least 90% identical to a sequence of at least 10 contiguous aminoacids in a sequence selected from the group consisting of: an amino acidsequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1A;and a complete amino acid sequence of a protein encoded by a human cDNAclone identified by a cDNA Clone Identifier in Table 1A and contained inthe deposit with the ATCC Deposit Number shown for said cDNA clone inTable 1A. Further, the step of comparing sequences in the above methodmay be performed by comparing the amino acid sequence determined from apolypeptide molecule in said sample with said sequence selected fromsaid group.

Also preferred is a method for identifying the species, tissue or celltype of a biological sample which method comprises a step of detectingpolypeptide molecules in said sample, if any, comprising an amino acidsequence that is at least 90% identical to a sequence of at least 10contiguous amino acids in a sequence selected from the group consistingof: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer asdefined in Table 1A; and a complete amino acid sequence of a secretedprotein encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table 1A and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1A. This method mayfurther comprise a step of detecting polypeptide molecules comprising anamino acid sequence in a panel of at least two amino acid sequences,wherein at least one sequence in said panel is at least 90% identical toa sequence of at least 10 contiguous amino acids in a sequence selectedfrom the above group.

Also preferred is a method for diagnosing in a subject a pathologicalcondition associated with abnormal structure or expression of a geneencoding a secreted protein identified in Table 1A, which methodcomprises a step of detecting in a biological sample obtained from saidsubject polypeptide molecules comprising an amino acid sequence in apanel of at least two amino acid sequences, wherein at least onesequence in said panel is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table 1A; and a complete amino acid sequence of asecreted protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table 1A and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1A.

In any of these methods, the step of detecting said polypeptidemolecules includes using an antibody.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a nucleotidesequence encoding a polypeptide wherein said polypeptide comprises anamino acid sequence that is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table 1A; and a complete amino acid sequence of asecreted protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table 1A and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table 1A. Further preferredis the above isolated nucleic acid molecule, wherein said nucleotidesequence encoding a polypeptide has been optimized for expression ofsaid polypeptide in a prokaryotic host. Similarly preferred is the aboveisolated nucleic acid molecule, wherein said polypeptide comprises anamino acid sequence selected from the group consisting of: an amino acidsequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1A;and a complete amino acid sequence of a secreted protein encoded by ahuman cDNA clone identified by a cDNA Clone Identifier in Table 1A andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table 1A.

Further preferred is a method of making a recombinant vector comprisinginserting any of the above isolated nucleic acid molecules into avector. Also preferred is the recombinant vector produced by thismethod. Also preferred is a method of making a recombinant host cellcomprising introducing the vector of the invention into a host cell, aswell as the recombinant host cell produced by this method.

Also preferred is a method of making an isolated polypeptide comprisingculturing this recombinant host cell under conditions such that saidpolypeptide is expressed and recovering said polypeptide. Also preferredis this method of making an isolated polypeptide, wherein saidrecombinant host cell is a eukaryotic cell and said polypeptide is asecreted portion of a human secreted protein comprising an amino acidsequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:Y beginning with the residue at the position of the FirstAmino Acid of the Secreted Portion of SEQ ID NO:Y wherein Y is aninteger set forth in Table 1A and said position of the First Amino Acidof the Secreted Portion of SEQ ID NO:Y is defined in Table 1A; and anamino acid sequence of a secreted portion of a protein encoded by ahuman cDNA clone identified by a cDNA Clone Identifier in Table 1A andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table 1A. The isolated polypeptide produced by this methodis also preferred.

Also preferred is a method of treatment of an individual in need of anincreased level of a secreted protein activity, which method comprisesadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated polypeptide, polynucleotide, orantibody of the claimed invention effective to increase the level ofsaid protein activity in said individual.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Isolation of a Selected cDNA Clone From the DepositedSample

Each cDNA clone in a cited ATCC deposit is contained in a plasmidvector. Table 1A identifies the vectors used to construct the cDNAlibrary from which each clone was isolated. In many cases, the vectorused to construct the library is a phage vector from which a plasmid hasbeen excised. The table immediately below correlates the related plasmidfor each phage vector used in constructing the cDNA library. Forexample, where a particular clone is identified in Table 1A as beingisolated in the vector “Lambda Zap,” the corresponding deposited cloneis in “pBluescript.”

Vector Used to Construct Library Corresponding Deposited Plasmid LambdaZap pBluescript (pBS) Uni-Zap XR pBluescript (pBS) Zap Express pBKlafmid BA plafmid BA pSport1 pSport1 pCMVSport 2.0 pCMVSport 2.0pCMVSport 3.0 pCMVSport 3.0 pCR ® 2.1 pCR ® 2.1

Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR(U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos.5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al.,Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J.M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. etal., Strategies 5:58-61 (1992)) are commercially available fromStratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla,Calif., 92037. pBS contains an ampicillin resistance gene and pBKcontains a neomycin resistance gene. Both can be transformed into E.coli strain XL-1 Blue, also available from Stratagene. pBS comes in 4forms SK+, SK−, KS+ and KS. The S and K refers to the orientation of thepolylinker to the T7 and T3 primer sequences which flank the polylinkerregion (“S” is for SacI and “K” is for KpnI which are the first sites oneach respective end of the linker). “+” or “−” refer to the orientationof the f1 origin of replication (“ori”), such that in one orientation,single stranded rescue initiated from the f1 ori generates sense strandDNA and in the other, antisense.

Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtained fromLife Technologies, Inc., P. O. Box 6009, Gaithersburg, Md. 20897. AllSport vectors contain an ampicillin resistance gene and may betransformed into E. coli strain DH10B, also available from LifeTechnologies. (See, for instance, Gruber, C. E., et al., Focus 15:59(1993).) Vector lafmid BA (Bento Soares, Columbia University, N.Y.)contains an ampicillin resistance gene and can be transformed into E.coli strain XL-1 Blue. Vector pCR®2.1, which is available fromInvitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains anampicillin resistance gene and may be transformed into E. coli strainDH10B, available from Life Technologies. (See, for instance, Clark, J.M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al.,Bio/Technology 9: (1991).) Preferably, a polynucleotide of the presentinvention does not comprise the phage vector sequences identified forthe particular clone in Table 1A, as well as the corresponding plasmidvector sequences designated above.

The deposited material in the sample assigned the ATCC Deposit Numbercited in Table 1A for any given cDNA clone also may contain one or moreadditional plasmids, each comprising a cDNA clone different from thatgiven clone. Thus, deposits sharing the same ATCC Deposit Number containat least a plasmid for each cDNA clone identified in Table 1A.Typically, each ATCC deposit sample cited in Table 1A comprises amixture of approximately equal amounts (by weight) of about 50 plasmidDNAs, each containing a different cDNA clone; but such a deposit samplemay include plasmids for more or less than 50 cDNA clones, up to about500 cDNA clones.

Two approaches can be used to isolate a particular clone from thedeposited sample of plasmid DNAs cited for that clone in Table 1A.First, a plasmid is directly isolated by screening the clones using apolynucleotide probe corresponding to SEQ ID NO:X.

Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with³²P-γ-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmidmixture is transformed into a suitable host, as indicated above (such asXL-1 Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents cited above. The transformants are plated on1.5% agar plates (containing the appropriate selection agent, e.g.,ampicillin) to a density of about 150 transformants (colonies) perplate. These plates are screened using Nylon membranes according toroutine methods for bacterial colony screening (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold SpringHarbor Laboratory Press, pages 1.93 to 1.104), or other techniques knownto those of skill in the art.

Alternatively, two primers of 17-20 nucleotides derived from both endsof the SEQ ID NO:X (i.e., within the region of SEQ ID NO:X bounded bythe 5′ NT and the 3′ NT of the clone defined in Table 1A) aresynthesized and used to amplify the desired cDNA using the depositedcDNA plasmid as a template. The polymerase chain reaction is carried outunder routine conditions, for instance, in 25 ul of reaction mixturewith 0.5 ug of the above cDNA template. A convenient reaction mixture is1.5-5 MM MgCl₂, 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP,dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirtyfive cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at55 degree C. for 1 min; elongation at 72 degree C. for 1 min) areperformed with a Perkin-Elmer Cetus automated thermal cycler. Theamplified product is analyzed by agarose gel electrophoresis and the DNAband with expected molecular weight is excised and purified. The PCRproduct is verified to be the selected sequence by subcloning andsequencing the DNA product.

Several methods are available for the identification of the 5′ or 3′non-coding portions of a gene which may not be present in the depositedclone. These methods include but are not limited to, filter probing,clone enrichment using specific probes, and protocols similar oridentical to 5′ and 3′ “RACE” protocols which are well known in the art.For instance, a method similar to 5′ RACE is available for generatingthe missing 5′ end of a desired full-length transcript. (Fromont-Racineet al., Nucleic Acids Res. 21(7):1683-1684 (1993).)

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full length gene.

This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA which may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strandcDNA synthesis using a gene specific oligonucleotide. The first strandsynthesis reaction is used as a template for PCR amplification of thedesired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene.

Example 2 Isolation of Genomic Clones Corresponding to a Polynucleotide

A human genomic P1 library (Genomic Systems, Inc.) is screened by PCRusing primers selected for the cDNA sequence corresponding to SEQ IDNO:X., according to the method described in Example 1. (See also,Sambrook.)

Example 3 Tissue Distribution of Polypeptide

Tissue distribution of mRNA expression of polynucleotides of the presentinvention is determined using protocols for Northern blot analysis,described by, among others, Sambrook et al. For example, a cDNA probeproduced by the method described in Example 1 is labeled with P³² usingthe rediprime™ DNA labeling system (Amersham Life Science), according tomanufacturer's instructions. After labeling, the probe is purified usingCHROMA SPIN-100™ column (Clontech Laboratories, Inc.), according tomanufacturer's protocol number PT1200-1. The purified labeled probe isthen used to examine various human tissues for mRNA expression.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) (Clontech) are examined with thelabeled probe using ExpressHyb™ hybridization solution (Clontech)according to manufacturer's protocol number PT1190-1. Followinghybridization and washing, the blots are mounted and exposed to film at−70 degree C. overnight, and the films developed according to standardprocedures.

Example 4 Chromosomal Mapping of the Polynucleotides

An oligonucleotide primer set is designed according to the sequence atthe 5′ end of SEQ ID NO:X. This primer preferably spans about 100nucleotides. This primer set is then used in a polymerase chain reactionunder the following set of conditions: 30 seconds, 95 degree C.; 1minute, 56 degree C.; 1 minute, 70 degree C. This cycle is repeated 32times followed by one 5 minute cycle at 70 degree C. Human, mouse, andhamster DNA is used as template in addition to a somatic cell hybridpanel containing individual chromosomes or chromosome fragments (Bios,Inc). The reactions is analyzed on either 8% polyacrylamide gels or 3.5%agarose gels. Chromosome mapping is determined by the presence of anapproximately 100 bp PCR fragment in the particular somatic cell hybrid.

Example 5 Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 1, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI, at the 5′end of the primers in order to clone the amplified product into theexpression vector. For example, BamHI and XbaI correspond to therestriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter/operator (P/O), a ribosome bindingsite (RBS), a 6-histidine tag (6-His), and restriction enzyme cloningsites.

The pQE-9 vector is digested with BamHI and XbaI and the amplifiedfragment is ligated into the pQE-9 vector maintaining the reading frameinitiated at the bacterial RBS. The ligation mixture is then used totransform the E. coli strain M15/rep4 (Qiagen, Inc.) which containsmultiple copies of the plasmid pREP4, which expresses the lacI repressorand also confers kanamycin resistance (Kan^(r)). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG inducesby inactivating the lacI repressor, clearing the P/O leading toincreased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000×g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at4 degree C. The cell debris is removed by centrifugation, and thesupernatant containing the polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6× His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl,pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl,pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finallythe polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4 degree C. or frozen at −80 degree C.

In addition to the above expression vector, the present inventionfurther includes an expression vector comprising phage operator andpromoter elements operatively linked to a polynucleotide of the presentinvention, called pHE4a. (ATCC Accession Number 209645, deposited onFeb. 25, 1998.) This vector contains: 1) a neomycinphosphotransferasegene as a selection marker, 2) an E. coli origin of replication, 3) a T5phage promoter sequence, 4) two lac operator sequences, 5) aShine-Delgarno sequence, and 6) the lactose operon repressor gene(lacIq). The origin of replication (oriC) is derived from pUC19 (LTI,Gaithersburg, Md.). The promoter sequence and operator sequences aremade synthetically.

DNA can be inserted into the pHEa by restricting the vector with NdeIand XbaI, BamHI, XhoI, or Asp718, running the restricted product on agel, and isolating the larger fragment (the stuffer fragment should beabout 310 base pairs). The DNA insert is generated according to the PCRprotocol described in Example 1, using PCR primers having restrictionsites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer).The PCR insert is gel purified and restricted with compatible enzymes.The insert and vector are ligated according to standard protocols.

The engineered vector could easily be substituted in the above protocolto express protein in a bacterial system.

Example 6 Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptideexpressed in E coli when it is present in the form of inclusion bodies.Unless otherwise specified, all of the following steps are conducted at4-10 degree C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10 degree C. and the cells harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4 degree C. without mixing for 12 hoursprior to further purification steps.

To clarify the refolded polypeptide solution, a previously preparedtangential filtration unit equipped with 0.16 um membrane filter withappropriate surface area (e.g., Filtron), equilibrated with 40 mM sodiumacetate, pH 6.0 is employed. The filtered sample is loaded onto a cationexchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column iswashed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM,1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. Theabsorbance at 280 nm of the effluent is continuously monitored.Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4volumes of water. The diluted sample is then loaded onto a previouslyprepared set of tandem columns of strong anion (Poros HQ-50, PerseptiveBiosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchangeresins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0.Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl.The CM-20 column is then eluted using a 10 column volume linear gradientranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50mM sodium acetate, pH 6.5. Fractions are collected under constant A₂₈₀monitoring of the effluent. Fractions containing the polypeptide(determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity afterthe above refolding and purification steps. No major contaminant bandsshould be observed from Commassie blue stained 16% SDS-PAGE gel when 5ug of purified protein is loaded. The purified protein can also betested for endotoxin/LPS contamination, and typically the LPS content isless than 0.1 ng/ml according to LAL assays.

Example 7 Cloning and Expression of a Polypeptide in a BaculovirusExpression System

In this example, the plasmid shuttle vector pA2 is used to insert apolynucleotide into a baculovirus to express a polypeptide. Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI, Xba I and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate a viable virus thatexpress the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above,such as pAc373, pVL941, and pAcIM1, as one skilled in the art wouldreadily appreciate, as long as the construct provides appropriatelylocated signals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

Specifically, the cDNA sequence contained in the deposited clone,including the AUG initiation codon and the naturally associated leadersequence identified in Table 1A, is amplified using the PCR protocoldescribed in Example 1. If the naturally occurring signal sequence isused to produce the secreted protein, the pA2 vector does not need asecond signal peptide. Alternatively, the vector can be modified (pA2GP) to include a baculovirus leader sequence, using the standard methodsdescribed in Summers et al., “A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures,” Texas AgriculturalExperimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together withT4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such asXL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria containing the plasmid are identified by digesting DNA fromindividual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

Five ug of a plasmid containing the polynucleotide is co-transfectedwith 1.0 ug of a commercially available linearized baculovirus DNA(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virus DNA and 5 ugof the plasmid are mixed in a sterile well of a microtiter platecontaining 50 ul of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10.) After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf) The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus containing the polynucleotideat a multiplicity of infection (“MOI”) of about 2. If radiolabeledproteins are desired, 6 hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of³⁵S-methionine and 5 uCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe produced protein.

Example 8 Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammaliancell. A typical mammalian expression vector contains a promoter element,which mediates the initiation of transcription of mRNA, a protein codingsequence, and signals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription isachieved with the early and late promoters from SV40, the long terminalrepeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the earlypromoter of the cytomegalovirus (CMN). However, cellular elements canalso be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells thatcould be used include, human Hela, 293, H9 and Jurkat cells, mouseNIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse Lcells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell linescontaining the polynucleotide integrated into a chromosome. Theco-transfection with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transfectedcells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulin developing cell lines that carry several hundred or even severalthousand copies of the gene of interest. (See, e.g., Alt, F. W., et al.,J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem.et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A.,Biotechnology 9:64-68 (1991).) Another useful selection marker is theenzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279(1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using thesemarkers, the mammalian cells are grown in selective medium and the cellswith the highest resistance are selected. These cell lines contain theamplified gene(s) integrated into a chromosome. Chinese hamster ovary(CHO) and NSO cells are often used for the production of proteins.

Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), theexpression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCCAccession No. 209647) contain the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985).) Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors also contain the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene,and the mouse DHFR gene under control of the SV40 early promoter.

Specifically, the plasmid pC6, for example, is digested with appropriaterestriction enzymes and then dephosphorylated using calf intestinalphosphates by procedures known in the art. The vector is then isolatedfrom a 1% agarose gel.

A polynucleotide of the present invention is amplified according to theprotocol outlined in Example 1. If the naturally occurring signalsequence is used to produce the secreted protein, the vector does notneed a second signal peptide. Alternatively, if the naturally occurringsignal sequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/348911.)

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzymeand purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used fortransfection. Five μg of the expression plasmid pC6 a pC4 iscotransfected with 0.5 ug of the plasmid pSVneo using lipofectin(Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the neo gene from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days singleclones are trypsinized and then seeded in 6-well petri dishes or 10 mlflasks using different concentrations of methotrexate (50 nM, 100 nM,200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM).The same procedure is repeated until clones are obtained which grow at aconcentration of 100-200 uM. Expression of the desired gene product isanalyzed, for instance, by SDS-PAGE and Western blot or by reversedphase HPLC analysis.

Example 9 Protein Fusions

The polypeptides of the present invention are preferably fused to otherproteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Example 5; see also EP A 394,827;Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion toIgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclearlocalization signals fused to the polypeptides of the present inventioncan target the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof a fusion protein. Fusion proteins can also create chimeric moleculeshaving more than one function. Finally, fusion proteins can increasesolubility and/or stability of the fused protein compared to thenon-fused protein. All of the types of fusion proteins described abovecan be made by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule, or the protocol described inExample 5.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector.

For example, if pC4 (Accession No. 209646) is used, the human Fc portioncan be ligated into the BamHI cloning site. Note that the 3′ BamHI siteshould be destroyed. Next, the vector containing the human Fc portion isre-restricted with BamHI, linearizing the vector, and a polynucleotideof the present invention, isolated by the PCR protocol described inExample 1, is ligated into this BamHI site. Note that the polynucleotideis cloned without a stop codon, otherwise a fusion protein will not beproduced.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

Human IgG Fc region: (SEQ ID NO:1)GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 10 Production of an Antibody from a Polypeptide

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing a polypeptide of the present invention isadministered to an animal to induce the production of sera containingpolyclonal antibodies. In a preferred method, a preparation of thesecreted protein is prepared and purified to render it substantiallyfree of natural contaminants. Such a preparation is then introduced intoan animal in order to produce polyclonal antisera of greater specificactivity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or protein binding fragments thereof). Suchmonoclonal

antibodies can be prepared using hybridoma technology. (Köhler et al.,Nature 256:495 (1975); Köhler et al., Eur. J. Immunol. 6:511 (1976);Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681(1981).) In general, such procedures involve immunizing an animal(preferably a mouse) with polypeptide or, more preferably, with asecreted polypeptide-expressing cell. Such cells may be cultured in anysuitable tissue culture medium; however, it is preferable to culturecells in Earle's modified Eagle's medium supplemented with 10% fetalbovine serum (inactivated at about 56 degrees C.), and supplemented withabout 10 g/l of nonessential amino acids, about 1,000 U/ml ofpenicillin, and about 100 ug/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP2O), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the polypeptide.

Alternatively, additional antibodies capable of binding to thepolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the protein-specificantibody can be blocked by the polypeptide. Such antibodies compriseanti-idiotypic antibodies to the protein-specific antibody and can beused to immunize an animal to induce formation of furtherprotein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, secretedprotein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).)

Example 11 Production Of Secreted Protein For High-Throughput ScreeningAssays

The following protocol produces a supernatant containing a polypeptideto be tested. This supernatant can then be used in the Screening Assaysdescribed herein.

First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution(1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516FBiowhittaker) for a working solution of 50 ug/ml. Add 200 ul of thissolution to each well (24 well plates) and incubate at RT for 20minutes. Be sure to distribute the solution over each well (note: a12-channel pipetter may be used with tips on every other channel).Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS(Phosphate Buffered Saline). The PBS should remain in the well untiljust prior to plating the cells and plates may be poly-lysine coated inadvance for up to two weeks.

Plate 293T cells (do not carry cells past P+20) at 2×10⁵ cells/well in0.5 ml DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose andL-glutamine (12-604F Biowhittaker))/10% heat inactivated FBS(14-503FBiowhittaker)/1× Penstrep(17-602E Biowhittaker). Let the cells growovernight.

The next day, mix together in a sterile solution basin: 300 ulLipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter,aliquot approximately 2 ug of an expression vector containing apolynucleotide insert, produced by the methods described in Examples 8or 9, into an appropriately labeled 96-well round bottom plate. With amulti-channel pipetter, add 50 ul of the Lipofectamine/Optimem I mixtureto each well. Pipette up and down gently to mix. Incubate at RT 15-45minutes. After about 20 minutes, use a multi-channel pipetter to add 150ul Optimem I to each well. As a control, one plate of vector DNA lackingan insert should be transfected with each set of transfections.

Preferably, the transfection should be performed by tag-teaming thefollowing tasks. By tag-teaming, hands on time is cut in half, and thecells do not spend too much time on PBS. First, person A aspirates offthe media from four 24-well plates of cells, and then person B rinseseach well with 0.5-1 ml PBS. Person A then aspirates off PBS rinse, andperson B, using a 12-channel pipetter with tips on every other channel,adds the 200 ul of DNA/Lipofectamine/Optimem I complex to the odd wellsfirst, then to the even wells, to each row on the 24-well plates.Incubate at 37 degrees C. for 6 hours.

While cells are incubating, prepare appropriate media, either 1% BSA inDMEM with 1× penstrep, or CHO-5 media (116.6 mg/L of CaCl2 (anhyd);0.00130 mg/L CuSO₄-5H₂O; 0.050 mg/L of Fe(NO₃)₃-9H₂O; 0.417 mg/L ofFeSO₄-7H₂O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl₂; 48.84 mg/L ofMgSO₄; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO₃; 62.50 mg/L ofNaH₂PO₄-H₂0; 71.02 mg/L of Na₂HPO4; 0.4320 mg/L of ZnSO₄-7H₂O; 0.002mg/L of Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L ofDL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L ofLinolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid;0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L ofPluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml ofL-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂0; 6.65 mg/ml of L-AsparticAcid; 29.56 mg/ml of L-Cystine-2HCL-H₂0; 31.29 mg/ml of L-Cystine-2HCL;7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/mlof Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂0; 106.97 mg/ml ofL-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL;32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/mlof L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H₂0; 99.65mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-CaPantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid;15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L ofPyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin;3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; and 0.680 mg/L ofVitamin B₁₂; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L ofSodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM of Ethanolamine;0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrincomplexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrincomplexed with Oleic Acid; and 10 mg/L of Methyl-B-Cyclodextrincomplexed with Retinal) with 2 mm glutamine and 1× penstrep. (BSA(81-068-3 Bayer) 100 gm dissolved in 1L DMEM for a 10% BSA stocksolution). Filter the media and collect 50 ul for endotoxin assay in 15ml polystyrene conical.

The transfection reaction is terminated, preferably by tag-teaming, atthe end of the incubation period. Person A aspirates off thetransfection media, while person B adds 1.5 ml appropriate media to eachwell. Incubate at 37 degrees C. for 45 or 72 hours depending on themedia used: 1% BSA for 45 hours or CHO-5 for 72 hours.

On day four, using a 300 ul multichannel pipetter, aliquot 600 ul in one1 ml deep well plate and the remaining supernatant into a 2 ml deepwell. The supernatants from each well can then be used in the assaysdescribed in Examples 13-20.

It is specifically understood that when activity is obtained in any ofthe assays described below using a supernatant, the activity originatesfrom either the polypeptide directly (e.g., as a secreted protein) or bythe polypeptide inducing expression of other proteins, which are thensecreted into the supernatant. Thus, the invention further provides amethod of identifying the protein in the supernatant characterized by anactivity in a particular assay.

Example 12 Construction of GAS Reporter Construct

One signal transduction pathway involved in the differentiation andproliferation of cells is called the Jaks-STATs pathway. Activatedproteins in the Jaks-STATs pathway bind to gamma activation site “GAS”elements or interferon-sensitive responsive element (“ISRE”), located inthe promoter of many genes. The binding of a protein to these elementsalter the expression of the associated gene.

GAS and ISRE elements are recognized by a class of transcription factorscalled Signal Transducers and Activators of Transcription, or “STATs.”There are six members of the STATs family. Stat1 and Stat3 are presentin many cell types, as is Stat2 (as response to IFN-alpha iswidespread). Stat4 is more restricted and is not in many cell typesthough it has been found in T helper class I, cells after treatment withIL-12. Stat5 was originally called mammary growth factor, but has beenfound at higher concentrations in other cells including myeloid cells.It can be activated in tissue culture cells by many cytokines.

The STATs are activated to translocate from the cytoplasm to the nucleusupon tyrosine phosphorylation by a set of kinases known as the JanusKinase (“Jaks”) family. Jaks represent a distinct family of solubletyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinasesdisplay significant sequence similarity and are generally catalyticallyinactive in resting cells.

The Jaks are activated by a wide range of receptors summarized in theTable below. (Adapted from review by Schidler and Darnell, Ann. Rev.Biochem. 64:621-51 (1995).) A cytokine receptor family, capable ofactivating Jaks, is divided into two groups: (a) Class 1 includesreceptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15,Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share aconserved cysteine motif (a set of four conserved cysteines and onetryptophan) and a WSXWS motif (a membrane proximal region encodingTrp-Ser-Xxx-Trp-Ser (SEQ ID NO:2)).

Thus, on binding of a ligand to a receptor, Jaks are activated, which inturn activate STATs, which then translocate and bind to GAS elements.This entire process is encompassed in the Jaks-STATs signal transductionpathway.

Therefore, activation of the Jaks-STATs pathway, reflected by thebinding of the GAS or the ISRE element, can be used to indicate proteinsinvolved in the proliferation and differentiation of cells. For example,growth factors and cytokines are known to activate the Jaks-STATspathway. (See Table below.) Thus, by using GAS elements linked toreporter molecules, activators of the Jaks-STATs pathway can beidentified.

JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS (elements) or ISRE IFN familyIFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 > IFP)Il-10 + ? ? − 1, 3 gp130 family IL-6 (Pleiotropic) + + + ? 1, 3 GAS(IRF1 > Lys6 > IFP) Il-11 (Pleiotropic) ? + ? ? 1, 3 OnM (Pleiotropic)? + + ? 1, 3 LIF (Pleiotropic) ? + + ? 1, 3 CNTF (Pleiotropic) −/+ + + ?1, 3 G-CSF (Pleiotropic) ? + ? ? 1, 3 IL-12 (Pleiotropic) + − + + 1, 3g-C family IL-2 (lymphocytes) − + − + 1, 3, 5 GAS IL-4 (lymph/myeloid)− + − + 6 GAS (IRF1 = IFP >> Ly6) (IgH) IL-7 (lymphocytes) − + − + 5 GASIL-9 (lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15? + ? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >>Ly6) IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growthhormone family GH ? − + − 5 PRL ? +/− + − 1, 3, 5 EPO ? − + − 5 GAS(B-CAS > LRF1 = IFP >> Ly6) Receptor Tyrosine Kinases EGF ? + + − 1, 3GAS (IRF1) PDGF ? + + − 1, 3 CSF-1 ? + + − 1, 3 GAS (not IRF1)

To construct a synthetic GAS containing promoter element, which is usedin the Biological Assays described in Examples 13-14, a PCR basedstrategy is employed to generate a GAS-SV40 promoter sequence. The 5′primer contains four tandem copies of the GAS binding site found in theIRF1 promoter and previously demonstrated to bind STATs upon inductionwith a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).),although other GAS or ISRE elements can be used instead. The 5′ primeralso contains 18 bp of sequence complementary to the SV40 early promotersequence and is flanked with an XhoI site. The sequence of the 5′ primeris:

(SEQ ID NO:3) 5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3′

The downstream primer is complementary to the SV40 promoter and isflanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ IDNO:4)

PCR amplification is performed using the SV40 promoter template presentin the B-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI/Hind III and subcloned into BLSK2-.(Stratagene.) Sequencing with forward and reverse primers confirms thatthe insert contains the following sequence:

(SEQ ID NO:5) 5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3′

With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2reporter construct is next engineered. Here, the reporter molecule is asecreted alkaline phosphatase, or “SEAP.” Clearly, however, any reportermolecule can be instead of SEAP, in this or in any of the otherExamples. Well known reporter molecules that can be used instead of SEAPinclude chloramphenicol acetyltransferase (CAT), luciferase, alkalinephosphatase, B-galactosidase, green fluorescent protein (GFP), or anyprotein detectable by an antibody.

The above sequence confirmed synthetic GAS-SV40 promoter element issubcloned into the pSEAP-Promoter vector obtained from Clontech usingHindIII and XhoI, effectively replacing the SV40 promoter with theamplified GAS:SV40 promoter element, to create the GAS-SEAP vector.However, this vector does not contain a neomycin resistance gene, andtherefore, is not preferred for mammalian expression systems.

Thus, in order to generate mammalian stable cell lines expressing theGAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAPvector using SalI and NotI, and inserted into a backbone vectorcontaining the neomycin resistance gene, such as pGFP-1 (Clontech),using these restriction sites in the multiple cloning site, to createthe GAS-SEAP/Neo vector. Once this vector is transfected into mammaliancells, this vector can then be used as a reporter molecule for GASbinding as described in Examples 13-14.

Other constructs can be made using the above description and replacingGAS with a different promoter sequence. For example, construction ofreporter molecules containing NFK-B and EGR promoter sequences aredescribed in Examples 15 and 16. However, many other promoters can besubstituted using the protocols described in these Examples. Forinstance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted,alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, II-2/NFAT, orNF-KB/GAS). Similarly, other cell lines can be used to test reporterconstruct activity, such as HELA (epithelial), HUVEC (endothelial), Reh(B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.

Example 13 High-Throughput Screening Assay for T-cell Activity

The following protocol is used to assess T-cell activity by identifyingfactors, and determining whether supernate containing a polypeptide ofthe invention proliferates and/or differentiates T-cells. T-cellactivity is assessed using the GAS/SEAP/Neo construct produced inExample 12. Thus, factors that increase SEAP activity indicate theability to activate the Jaks-STATS signal transduction pathway. TheT-cell used in this assay is Jurkat T-cells (ATCC Accession No.TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4cells (ATCC Accession No. CRL-1582) cells can also be used.

Jurkat T-cells are lymphoblastic CD4+Th1 helper cells. In order togenerate stable cell lines, approximately 2 million Jurkat cells aretransfected with the GAS-SEAP/neo vector using DMRIE-C (LifeTechnologies)(transfection procedure described below). The transfectedcells are seeded to a density of approximately 20,000 cells per well andtransfectants resistant to 1 mg/ml genticin selected. Resistant coloniesare expanded and then tested for their response to increasingconcentrations of interferon gamma. The dose response of a selectedclone is demonstrated.

Specifically, the following protocol will yield sufficient cells for 75wells containing 200 ul of cells. Thus, it is either scaled up, orperformed in multiple to generate sufficient cells for multiple 96 wellplates. Jurkat cells are maintained in RPMI+10% serum with 1% Pen-Strep.Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug of plasmidDNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C andincubate at room temperature for 15-45 mins.

During the incubation period, count cell concentration, spin down therequired number of cells (10⁷ per transfection), and resuspend inOPTI-MEM to a final concentration of 10⁷ cells/ml. Then add 1 ml of1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37 degrees C. for 6hrs. After the incubation, add 10 ml of RPMI+15% serum.

The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10%serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated withsupernatants containing polypeptides of the invention and/or inducedpolypeptides of the invention as produced by the protocol described inExample 11.

On the day of treatment with the supernatant, the cells should be washedand resuspended in fresh RPMI+10% serum to a density of 500,000 cellsper ml. The exact number of cells required will depend on the number ofsupernatants being screened. For one 96 well plate, approximately 10million cells (for 10 plates, 100 million cells) are required.

Transfer the cells to a triangular reservoir boat, in order to dispensethe cells into a 96 well dish, using a 12 channel pipette. Using a 12channel pipette, transfer 200 ul of cells into each well (thereforeadding 100,000 cells per well).

After all the plates have been seeded, 50 ul of the supernatants aretransferred directly from the 96 well plate containing the supernatantsinto each well using a 12 channel pipette. In addition, a dose ofexogenous interferon gamma (0.1, 1.0, 10 ng) is added to wells H9, H10,and H11 to serve as additional positive controls for the assay.

The 96 well dishes containing Jurkat cells treated with supernatants areplaced in an incubator for 48 hrs (note: this time is variable between48-72 hrs). 35 ul samples from each well are then transferred to anopaque 96 well plate using a 12 channel pipette. The opaque platesshould be covered (using sellophene covers) and stored at −20 degrees C.until SEAP assays are performed according to Example 17. The platescontaining the remaining treated cells are placed at 4 degrees C. andserve as a source of material for repeating the assay on a specific wellif desired.

As a positive control, 100 Unit/ml interferon gamma can be used which isknown to activate Jurkat T cells. Over 30 fold induction is typicallyobserved in the positive control wells.

The above protocol may be used in the generation of both transient, aswell as, stable transfected cells, which would be apparent to those ofskill in the art.

Example 14 High-Throughput Screening Assay Identifying Myeloid Activity

The following protocol is used to assess myeloid activity by determiningwhether polypeptides of the invention proliferates and/or differentiatesmyeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neoconstruct produced in Example 12. Thus, factors that increase SEAPactivity indicate the ability to activate the Jaks-STATS signaltransduction pathway. The myeloid cell used in this assay is U937, apre-monocyte cell line, although TF-1, HL60, or KG1 can be used.

To transiently transfect U937 cells with the GAS/SEAP/Neo constructproduced in Example 12, a DEAE-Dextran method (Kharbanda et. al., 1994,Cell Growth & Differentiation, 5:259-265) is used. First, harvest 2×10e⁷U937 cells and wash with PBS. The U937 cells are usually grown in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS)supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.

Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffercontaining 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mMNaCl, 5 mM KCl, 375 uM Na₂HPO₄.7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂.Incubate at 37 degrees C. for 45 min.

Wash the cells with RPMI 1640 medium containing 10% FBS and thenresuspend in 10 ml complete medium and incubate at 37 degrees C. for 36hr.

The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400ug/ml G418. The G418-free medium is used for routine growth but everyone to two months, the cells should be re-grown in 400 ug/ml G418 forcouple of passages.

These cells are tested by harvesting 1×10⁸ cells (this is enough for ten96-well plates assay) and wash with PBS. Suspend the cells in 200 mlabove described growth medium, with a final density of 5×10⁵ cells/ml.Plate 200 ul cells per well in the 96-well plate (or 1×10⁵ cells/well).

Add 50 ul of the supernatant prepared by the protocol described inExample 11. Incubate at 37 degrees C. for 48 to 72 hr. As a positivecontrol, 100 Unit/ml interferon gamma can be used which is known toactivate U937 cells. Over 30 fold induction is typically observed in thepositive control wells. SEAP assay the supernatant according to theprotocol described in Example 17.

Example 15 High-Throughput Screening Assay Identifying Neuronal Activity

When cells undergo differentiation and proliferation, a group of genesare activated through many different signal transduction pathways. Oneof these genes, EGR1 (early growth response gene 1), is induced invarious tissues and cell types upon activation. The promoter of EGR1 isresponsible for such induction. Using the EGR1 promoter linked toreporter molecules, activation of cells can be assessed.

Particularly, the following protocol is used to assess neuronal activityin PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are knownto proliferate and/or differentiate by activation with a number ofmitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growthfactor), and EGF (epidermal growth factor). The EGR1 gene expression isactivated during this treatment. Thus, by stably transfecting PC12 cellswith a construct containing an EGR promoter linked to SEAP reporter,activation of PC12 cells can be assessed.

The EGR/SEAP reporter construct can be assembled by the followingprotocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al.,Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNAusing the following primers:

(SEQ ID NO:6) 5′ GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′ (SEQ ID NO:7)5′ GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′

Using the GAS:SEAP/Neo vector produced in Example 12, EGR1 amplifiedproduct can then be inserted into this vector. Linearize theGAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing theGAS/SV40 stuffer. Restrict the EGR1 amplified product with these sameenzymes. Ligate the vector and the EGR1 promoter.

To prepare 96 well-plates for cell culture, two mls of a coatingsolution (1:30 dilution of collagen type I (Upstate Biotech Inc.Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cmplate or 50 ml per well of the 96-well plate, and allowed to air dry for2 hr.

PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker)containing 10% horse serum (JRH BIOSCIENCES, Cat. #12449-78P), 5%heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/mlpenicillin and 100 ug/ml streptomycin on a precoated 10 cm tissueculture dish. One to four split is done every three to four days. Cellsare removed from the plates by scraping and resuspended with pipettingup and down for more than 15 times.

Transfect the EGR/SEAP/Neo construct into PC12 using the Lipofectamineprotocol described in Example 11. EGR-SEAP/PC12 stable cells areobtained by growing the cells in 300 ug/ml G418. The G418-free medium isused for routine growth but every one to two months, the cells should bere-grown in 300 ug/ml G418 for couple of passages.

To assay for neuronal activity, a 10 cm plate with cells around 70 to80% confluent is screened by removing the old medium. Wash the cellsonce with PBS (Phosphate buffered saline). Then starve the cells in lowserum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS withantibiotics) overnight.

The next morning, remove the medium and wash the cells with PBS. Scrapeoff the cells from the plate, suspend the cells well in 2 ml low serummedium. Count the cell number and add more low serum medium to reachfinal cell density as 5×10⁵ cells/ml.

Add 200 ul of the cell suspension to each well of 96-well plate(equivalent to 1×10⁵ cells/well). Add 50 ul supernatant produced byExample 11, 37° C. for 48 to 72 hr. As a positive control, a growthfactor known to activate PC12 cells through EGR can be used, such as 50ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAPis typically seen in the positive control wells. SEAP assay thesupernatant according to Example 17.

Example 16 High-Throughput Screening Assay for T-cell Activity

NF-KB (Nuclear Factor KB) is a transcription factor activated by a widevariety of agents including the inflammatory cytokines IL-1 and TNF,CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure toLPS or thrombin, and by expression of certain viral gene products. As atranscription factor, NF-KB regulates the expression of genes involvedin immune cell activation, control of apoptosis (NF-KB appears to shieldcells from apoptosis), B and T-cell development, anti-viral andantimicrobial responses, and multiple stress responses.

In non-stimulated conditions, NF-KB is retained in the cytoplasm withI-KB (Inhibitor KB). However, upon stimulation, I-KB is phosphorylatedand degraded, causing NF-KB to shuttle to the nucleus, therebyactivating transcription of target genes. Target genes activated byNF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

Due to its central role and ability to respond to a range of stimuli,reporter constructs utilizing the NF-KB promoter element are used toscreen the supernatants produced in Example 11. Activators or inhibitorsof NF-KB would be useful in treating diseases. For example, inhibitorsof NF-KB could be used to treat those diseases related to the acute orchronic activation of NF-KB, such as rheumatoid arthritis.

To construct a vector containing the NF-KB promoter element, a PCR basedstrategy is employed. The upstream primer contains four tandem copies ofthe NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO:8), 18 bp of sequencecomplementary to the 5′ end of the SV40 early promoter sequence, and isflanked with an XhoI site:

(SEQ ID NO:9) 5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCCTGCCATCTCAATTAG:3′

The downstream primer is complementary to the 3′ end of the SV40promoter and is flanked with a Hind III site:

5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO:4)

PCR amplification is performed using the SV40 promoter template presentin the pB-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI and Hind III and subcloned into BLSK2-.(Stratagene) Sequencing with the T7 and T3 primers confirms the insertcontains the following sequence:

(SEQ ID NO:10) 5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGC AAAAAGCTT:3′

Next, replace the SV40 minimal promoter element present in thepSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment usingXhoI and HindIII. However, this vector does not contain a neomycinresistance gene, and therefore, is not preferred for mammalianexpression systems.

In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAPcassette is removed from the above NF-KB/SEAP vector using restrictionenzymes SalI and NotI, and inserted into a vector containing neomycinresistance. Particularly, the NF-KB/SV40/SEAP cassette was inserted intopGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 withSalI and NotI.

Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells arecreated and maintained according to the protocol described in Example13. Similarly, the method for assaying supernatants with these stableJurkat T-cells is also described in Example 13. As a positive control,exogenous TNF alpha (0.1,1, 10 ng) is added to wells H9, H10, and H11,with a 5-10 fold activation typically observed.

Example 17 Assay for SEAP Activity

As a reporter molecule for the assays described in Examples 13-16, SEAPactivity is assayed using the Tropix Phospho-light Kit (Cat. BP-400)according to the following general procedure. The Tropix Phospho-lightKit supplies the Dilution, Assay, and Reaction Buffers used below.

Prime a dispenser with the 2.5× Dilution Buffer and dispense 15 ul of2.5× dilution buffer into Optiplates containing 35 ul of a supernatant.Seal the plates with a plastic sealer and incubate at 65 degree C. for30 min. Separate the Optiplates to avoid uneven heating.

Cool the samples to room temperature for 15 minutes. Empty the dispenserand prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate atroom temperature 5 min. Empty the dispenser and prime with the ReactionBuffer (see the table below). Add 50 ul Reaction Buffer and incubate atroom temperature for 20 minutes. Since the intensity of thechemiluminescent signal is time dependent, and it takes about 10 minutesto read 5 plates on luminometer, one should treat 5 plates at each timeand start the second set 10 minutes later.

Read the relative light unit in the luminometer. Set H12 as blank, andprint the results. An increase in chemiluminescence indicates reporteractivity.

Reaction Buffer Formulation: # of plates Rxn buffer diluent (ml) CSPD(ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 16 904.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22 120 6 23125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.5 29 1557.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 185 9.25 36190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 215 10.75 42220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 245 12.25 48250 12.5 49 255 12.75 50 260 13

Example 18 High-Throughput Screening Assay Identifying Changes in SmallMolecule Concentration and Membrane Permeability

Binding of a ligand to a receptor is known to alter intracellular levelsof small molecules, such as calcium, potassium, sodium, and pH, as wellas alter membrane potential. These alterations can be measured in anassay to identify supernatants which bind to receptors of a particularcell. Although the following protocol describes an assay for calcium,this protocol can easily be modified to detect changes in potassium,sodium, pH, membrane potential, or any other small molecule which isdetectable by a fluorescent probe.

The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) tomeasure changes in fluorescent molecules (Molecular Probes) that bindsmall molecules. Clearly, any fluorescent molecule detecting a smallmolecule can be used instead of the calcium fluorescent molecule, fluo-4(Molecular Probes, Inc.; catalog no. F-14202), used here.

For adherent cells, seed the cells at 10,000-20,000 cells/well in aCo-star black 96-well plate with clear bottom. The plate is incubated ina CO₂ incubator for 20 hours. The adherent cells are washed two times inBiotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution)leaving 100 ul of buffer after the final wash.

A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. Toload the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is added to eachwell. The plate is incubated at 37 degrees C. in a CO₂ incubator for 60min. The plate is washed four times in the Biotek washer with HBSSleaving 100 ul of buffer.

For non-adherent cells, the cells are spun down from culture media.Cells are re-suspended to 2-5×10⁶ cells/ml with HBSS in a 50-ml conicaltube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is addedto each ml of cell suspension. The tube is then placed in a 37 degreesC. water bath for 30-60 min. The cells are washed twice with HBSS,resuspended to 1×10⁶ cells/ml, and dispensed into a microplate, 100ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate isthen washed once in Denley CellWash with 200 ul, followed by anaspiration step to 100 ul final volume.

For a non-cell based assay, each well contains a fluorescent molecule,such as fluo-4. The supernatant is added to the well, and a change influorescence is detected.

To measure the fluorescence of intracellular calcium, the FLIPR is setfor the following parameters: (1) System gain is 300-800 mW; (2)Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul.Increased emission at 530 nm indicates an extracellular signaling eventwhich has resulted in an increase in the intracellular Ca⁺⁺concentration.

Example 19 High-Throughput Screening Assay Identifying Tyrosine KinaseActivity

The Protein Tyrosine Kinases (PTK) represent a diverse group oftransmembrane and cytoplasmic kinases. Within the Receptor ProteinTyrosine Kinase RPTK) group are receptors for a range of mitogenic andmetabolic growth factors including the PDGF, FGF, EGF, NGF, HGF andInsulin receptor subfamilies. In addition there are a large family ofRPTKs for which the corresponding ligand is unknown. Ligands for RPTKsinclude mainly secreted small proteins, but also membrane-bound andextracellular matrix proteins.

Activation of RPTK by ligands involves ligand-mediated receptordimerization, resulting in transphosphorylation of the receptor subunitsand activation of the cytoplasmic tyrosine kinases. The cytoplasmictyrosine kinases include receptor associated tyrosine kinases of thesrc-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked andcytosolic protein tyrosine kinases, such as the Jak family, members ofwhich mediate signal transduction triggered by the cytokine superfamilyof receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

Because of the wide range of known factors capable of stimulatingtyrosine kinase activity, the identification of novel human secretedproteins capable of activating tyrosine kinase signal transductionpathways are of interest. Therefore, the following protocol is designedto identify those novel human secreted proteins capable of activatingthe tyrosine kinase signal transduction pathways.

Seed target cells (e.g., primary keratinocytes) at a density ofapproximately 25,000 cells per well in a 96 well Loprodyne Silent ScreenPlates purchased from Nalge Nunc (Naperville, Ill.). The plates aresterilized with two 30 minute rinses with 100% ethanol, rinsed withwater and dried overnight. Some plates are coated for 2 hr with 100 mlof cell culture grade type I collagen (50 mg/ml), gelatin (2%) orpolylysine (50 mg/ml), all of which can be purchased from SigmaChemicals (St. Louis, Mo.) or 10% Matrigel purchased from BectonDickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at4 degree C. Cell growth on these plates is assayed by seeding 5,000cells/well in growth medium and indirect quantitation of cell numberthrough use of alamarBlue as described by the manufacturer AlamarBiosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers#3071 from Becton Dickinson (Bedford, Mass,) are used to cover theLoprodyne Silent Screen Plates. Falcon Microtest III cell culture platescan also be used in some proliferation experiments.

To prepare extracts, A431 cells are seeded onto the nylon membranes ofLoprodyne plates (20,000/200 ml/well) and cultured overnight in completemedium. Cells are quiesced by incubation in serum-free basal medium for24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or 50 ul of thesupernatant produced in Example 11, the medium was removed and 100 ml ofextraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100,0.1% SDS, 2 mM Na3VO4, 2 mM Na4P2O7 and a cocktail of proteaseinhibitors (#1836170) obtained from Boeheringer Mannheim (Indianapolis,Ind.) is added to each well and the plate is shaken on a rotating shakerfor 5 minutes at 4 degrees C. The plate is then placed in a vacuumtransfer manifold and the extract filtered through the 0.45 mm membranebottoms of each well using house vacuum. Extracts are collected in a96-well catch/assay plate in the bottom of the vacuum manifold andimmediately placed on ice. To obtain extracts clarified bycentrifugation, the content of each well, after detergent solubilizationfor 5 minutes, is removed and centrifuged for 15 minutes at 4 degrees C.at 16,000×g.

Test the filtered extracts for levels of tyrosine kinase activity.Although many methods of detecting tyrosine kinase activity are known,one method is described here.

Generally, the tyrosine kinase activity of a supernatant is evaluated bydetermining its ability to phosphorylate a tyrosine residue on aspecific substrate (a biotinylated peptide). Biotinylated peptides thatcan be used for this purpose include PSK1 (corresponding to amino acids6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding toamino acids 1-17 of gastrin). Both peptides are substrates for a rangeof tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction is set up by adding the followingcomponents in order. First, add 10 ul of 5 uM Biotinylated Peptide, then10 ul ATP/Mg₂₊ (5 mM ATP/50 mM MgCl₂), then 10 ul of 5× Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mMEGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then 5 ul of SodiumVanadate(1 mM), and then 5 ul of water. Mix the components gently andpreincubate the reaction mix at 30 degrees C. for 2 min. Initial thereaction by adding 10 ul of the control enzyme or the filteredsupernatant.

The tyrosine kinase assay reaction is then terminated by adding 10 ul of120 mm EDTA and place the reactions on ice.

Tyrosine kinase activity is determined by transferring 50 ul aliquot ofreaction mixture to a microtiter plate (MTP) module and incubating at 37degrees C. for 20 min. This allows the streptavadin coated 96 well plateto associate with the biotinylated peptide. Wash the MTP module with 300ul/well of PBS four times. Next add 75 ul of anti-phospotyrosineantibody conjugated to horse radish peroxidase(anti-P-Tyr-POD(0.5 u/ml))to each well and incubate at 37 degrees C. for one hour. Wash the wellas above.

Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim)and incubate at room temperature for at least 5 mins (up to 30 min).Measure the absorbance of the sample at 405 nm by using ELISA reader.The level of bound peroxidase activity is quantitated using an ELISAreader and reflects the level of tyrosine kinase activity.

Example 20 High-Throughput Screening Assay Identifying PhosphorylationActivity

As a potential alternative and/or compliment to the assay of proteintyrosine kinase activity described in Example 19, an assay which detectsactivation (phosphorylation) of major intracellular signal transductionintermediates can also be used. For example, as described below oneparticular assay can detect tyrosine phosphorylation of the Erk-1 andErk-2 kinases. However, phosphorylation of other molecules, such as Raf,JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specifickinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine,phosphotyrosine, or phosphothreonine molecule, can be detected bysubstituting these molecules for Erk-1 or Erk-2 in the following assay.

Specifically, assay plates are made by coating the wells of a 96-wellELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp,(RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBSfor 1 hr at RT. The protein G plates are then treated with 2 commercialmonoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT)(Santa Cruz Biotechnology). (To detect other molecules, this step caneasily be modified by substituting a monoclonal antibody detecting anyof the above described molecules.) After 3-5 rinses with PBS, the platesare stored at 4 degrees C. until use.

A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplateand cultured overnight in growth medium. The cells are then starved for48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50ul of the supernatants obtained in Example 11 for 5-20 minutes. Thecells are then solubilized and extracts filtered directly into the assayplate.

After incubation with the extract for 1 hr at RT, the wells are againrinsed. As a positive control, a commercial preparation of MAP kinase(10 ng/well) is used in place of A431 extract. Plates are then treatedwith a commercial polyclonal (rabbit) antibody (1 ug/ml) whichspecifically recognizes the phosphorylated epitope of the Erk-1 andErk-2 kinases (1 hr at RT). This antibody is biotinylated by standardprocedures. The bound polyclonal antibody is then quantitated bysuccessive incubations with Europium-streptavidin and Europiumfluorescence enhancing reagent in the Wallac DELFIA instrument(time-resolved fluorescence). An increased fluorescent signal overbackground indicates a phosphorylation.

Example 21 Method of Determining Alterations in a Gene Corresponding toa Polynucleotide

RNA isolated from entire families or individual patients presenting witha phenotype of interest (such as a disease) is be isolated. cDNA is thengenerated from these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:X. Suggested PCRconditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60-120seconds at 52-58 degrees C.; and 60-120 seconds at 70 degrees C., usingbuffer solutions described in Sidransky et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons isalso determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations is then cloned andsequenced to validate the results of the direct sequencing.

PCR products is cloned into T-tailed vectors as described in Holton etal., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7polymerase (United States Biochemical). Affected individuals areidentified by mutations not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in a gene corresponding to a polynucleotide. Genomic clonesisolated according to Example 2 are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson et al., Methods Cell Biol. 35:73-99(1991). Hybridization with the labeled probe is carried out using a vastexcess of human cot-1 DNA for specific hybridization to thecorresponding genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region hybridized by the probe are identifiedas insertions, deletions, and translocations. These alterations are usedas a diagnostic marker for an associated disease.

Example 22 Method of Detecting Abnormal Levels of a Polypeptide in aBiological Sample

A polypeptide of the present invention can be detected in a biologicalsample, and if an increased or decreased level of the polypeptide isdetected, this polypeptide is a marker for a particular phenotype.Methods of detection are numerous, and thus, it is understood that oneskilled in the art can modify the following assay to fit theirparticular needs.

For example, antibody-sandwich ELISAs are used to detect polypeptides ina sample, preferably a biological sample. Wells of a microtiter plateare coated with specific antibodies, at a final concentration of 0.2 to10 ug/ml. The antibodies are either monoclonal or polyclonal and areproduced by the method described in Example 10. The wells are blocked sothat non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining the polypeptide. Preferably, serial dilutions of the sampleshould be used to validate results. The plates are then washed threetimes with deionized or distilled water to remove unbounded polypeptide.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution to each well and incubate 1 hour atroom temperature. Measure the reaction by a microtiter plate reader.Prepare a standard curve, using serial dilutions of a control sample,and plot polypeptide concentration on the X-axis (log scale) andfluorescence or absorbance of the Y-axis (linear scale). Interpolate theconcentration of the polypeptide in the sample using the standard curve.

Example 23 Formulation

The invention also provides methods of treatment and/or prevention ofdiseases or disorders (such as, for example, any one or more of thediseases or disorders disclosed herein) by administration to a subjectof an effective amount of a Therapeutic. By therapeutic is meantpolynucleotides or polypeptides of the invention (including fragmentsand variants), agonists or antagonists thereof, and/or antibodiesthereto, in combination with a pharmaceutically acceptable carrier type(e.g., a sterile carrier).

The Therapeutic will be formulated and dosed in a fashion consistentwith good medical practice, taking into account the clinical conditionof the individual patient (especially the side effects of treatment withthe Therapeutic alone), the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofthe Therapeutic administered parenterally per dose will be in the rangeof about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although,as noted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the Therapeutic is typically administered at a dose rateof about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injectionsper day or by continuous subcutaneous infusions, for example, using amini-pump. An intravenous bag solution may also be employed. The lengthof treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

Therapeutics can be are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any. The term “parenteral” as usedherein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

In a preferred embodiment, Neutrokine-alpha and/or Neutrokine-alphaSVcompositions of the invention are formulated in a biodegradable,polymeric drug delivery system, for example as described in U.S. Pat.Nos. 4,938,763; 5,278,201; 5,278,202; 5,324,519; 5,340,849; and5,487,897 and in International Publication Numbers WO01/35929,WO00/24374, and WO00/06117 which are hereby incorporated by reference intheir entirety. In specific preferred embodiments the Neutrokine-alphaand/or Neutrokine-alphaSV compositions of the invention are formulatedusing the ATRIGEL® Biodegradable System of Atrix Laboratories, Inc.(Fort Collins, Colo.).

Examples of biodegradable polymers which can be used in the formulationof Neutrokine-alpha and/or Neutrokine-alphaSV compositions, include butare not limited to, polylactides, polyglycolides, polycaprolactones,polyanhydrides, polyamides, polyurethanes, polyesteramides,polyorthoesters, polydioxanones, polyacetals, polyketals,polycarbonates, polyorthocarbonates, polyphosphazenes,polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates,polyalkylene succinates, poly(malic acid), poly(amino acids),poly(methyl vinyl ether), poly(maleic anhydride), polyvinylpyrrolidone,polyethylene glycol, polyhydroxycellulose, chitin, chitosan, andcopolymers, terpolymers, or combinations or mixtures of the abovematerials. The preferred polymers are those that have a lower degree ofcrystallization and are more hydrophobic. These polymers and copolymersare more soluble in the biocompatible solvents than the highlycrystalline polymers such as polyglycolide and chitin which also have ahigh degree of hydrogen-bonding. Preferred materials with the desiredsolubility parameters are the polylactides, polycaprolactones, andcopolymers of these with glycolide in which there are more amorphousregions to enhance solubility. In specific preferred embodiments, thebiodegradable polymers which can be used in the formulation ofNeutrokine-alpha and/or Neutrokine-alphaSV compositions arepoly(lactide-co-glycolides). Polymer properties such as molecularweight, hydrophobicity, and lactide/glycolide ratio may be modified toobtain the desired drug Neutrokine-alpha and/or Neutrokine-alphaSVrelease profile (See, e.g., Ravivarapu et al., Journal of PharmaceuticalSciences 89:732-741 (2000), which is hereby incorporated by reference inits entirety).

It is also preferred that the solvent for the biodegradable polymer benon-toxic, water miscible, and otherwise biocompatible. Examples of suchsolvents include, but are not limited to, N-methyl-2-pyrrolidone,2-pyrrolidone, C2 to C6 alkanols, C1 to C15 alchohols, dils, triols, andtetraols such as ethanol, glycerine propylene glycol, butanol; C3 to C15alkyl ketones such as acetone, diethyl ketone and methyl ethyl ketone;C3 to C15 esters such as methyl acetate, ethyl acetate, ethyl lactate;alkyl ketones such as methyl ethyl ketone, C1 to C15 amides such asdimethylformamide, dimethylacetamide and caprolactam; C3 to C20 etherssuch as tetrahydrofuran, or solketal; tweens, triacetin, propylenecarbonate, decylmethylsulfoxide, dimethyl sulfoxide, oleic acid,1-dodecylazacycloheptan-2-one, Other preferred solvents are benzylalchohol, benzyl benzoate, dipropylene glycol, tributyrin, ethyl oleate,glycerin, glycofural, isopropyl myristate, isopropyl palmitate, oleicacid, polyethylene glycol, propylene carbonate, and triethyl citrate.The most preferred solvents are

N-methyl-2-pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide, triacetin,and propylene carbonate because of the solvating ability and theircompatibility.

Additionally, formulations comprising Neutrokine-alpha and/orNeutrokine-alphaSV compositions and a biodegradable polymer may alsoinclude release-rate modification agents and/or pore-forming agents.Examples of release-rate modification agents include, but are notlimited to, fatty acids, triglycerides, other like hydrophobiccompounds, organic solvents, plasticizing compounds and hydrophiliccompounds. Suitable release rate modification agents include, forexample, esters of mono-, di-, and tricarboxylic acids, such as2-ethoxyethyl acetate, methyl acetate, ethyl acetate, diethyl phthalate,dimethyl phthalate, dibutyl phthalate, dimethyl adipate, dimethylsuccinate, dimethyl oxalate, dimethyl citrate, triethyl citrate, acetyltributyl citrate, acetyl triethyl citrate, glycerol triacetate,di(n-butyl) sebecate, and the like; polyhydroxy alcohols, such aspropylene glycol, polyethylene glycol, glycerin, sorbitol, and the like;fatty acids; triesters of glycerol, such as triglycerides, epoxidizedsoybean oil, and other epoxidized vegetable oils; sterols, such ascholesterol; alcohols, such as C.sub.6 -C.sub.12 alkanols,2-ethoxyethanol, and the like. The release rate modification agent maybe used singly or in combination with other such agents. Suitablecombinations of release rate modification agents include, but are notlimited to, glycerin/propylene glycol, sorbitol/glycerine, ethyleneoxide/propylene oxide, butylene glycol/adipic acid, and the like.Preferred release rate modification agents include, but are not limitedto, dimethyl citrate, triethyl citrate, ethyl heptanoate, glycerin, andhexanediol. Suitable pore-forming agents that may be used in the polymercomposition include, but are not limited to, sugars such as sucrose anddextrose, salts such as sodium chloride and sodium carbonate, polymerssuch as hydroxylpropylcellulose, carboxymethylcellulose, polyethyleneglycol, and polyvinylpyrrolidone. Solid crystals that will provide adefined pore size, such as salt or sugar, are preferred.

In specific preferred embodiments the Neutrokine-alpha and/orNeutrokine-alphaSV compositions of the invention are formulated usingthe BEMA™ BioErodible Mucoadhesive System, MCA™ MucoCutaneous AbsorptionSystem, SMP™ Solvent MicroParticle System, or BCP™ BioCompatible PolymerSystem of Atrix Laboratories, Inc. (Fort Collins, Colo.).

Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317-327 and 353-365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); E P 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalTherapeutic.

In yet an additional embodiment, the Therapeutics of the invention aredelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the Therapeutic isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeuticuniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

Therapeutics ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of theTherapeutics of the invention. Associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition, the Therapeutics may be employedin conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG (e.g., THERACYS®), MPL and nonviable prepartionsof Corynebacterium parvum. In a specific embodiment, Therapeutics of theinvention are administered in combination with alum. In another specificembodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, chemotherapeutic agents, antibiotics,steroidal and non-steroidal anti-inflammatories, conventionalimmunotherapeutic agents, and/or therapeutic treatments described below.Combinations may be administered either concomitantly, e.g., as anadmixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In certain embodiments, Therapeutics of the invention are administeredin combination with antiretroviral agents, nucleoside/nucleotide reversetranscriptase inhibitors (NRTIs), non-nucleoside reverse transcriptaseinhibitors (NNRTIs), and/or protease inhibitors (PIs). NRTIs that may beadministered in combination with the Therapeutics of the invention,include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™(didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudine/d4T),EPIVIR™ (lamivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). NNRTIsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith Therapeutics of the invention to treat AIDS and/or to prevent ortreat HIV infection.

Additional NRTIs include LODENOSINE™ (F-ddA; an acid-stable adenosineNRTI; Triangle/Abbott; COVIRACIL™ (emtricitabine/PTC; structurallyrelated to lamivudine (3TC) but with 3-to 10-fold greater activity invitro; Triangle/Abbott); dOTC (BCH-10652, also structurally related tolamivudine but retains activity against a substantial proportion oflamivudine-resistant isolates; Biochem Pharma); Adefovir (refusedapproval for anti-HIV therapy by FDA; Gilead Sciences); PREVEON®(Adefovir Dipivoxil, the active prodrug of adefovir; its active form isPMEA-pp); TENOFOVIR™ (bis-POC PMPA, a PMPA prodrug; Gilead); DAPD/DXG(active metabolite of DAPD; Triangle/Abbott); D-D4FC (related to 3TC,with activity against AZT/3TC-resistant virus); GW420867X (GlaxoWellcome); ZIAGEN™ (abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3′azido-2′,3′-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl(SATE)-bearing prodrug forms of β-L-FD4C and β-L-FddC (WO 98/17281).

Additional NNRTIs include COACTINON™ (Emivirine/MKC-442, potent NNRTI ofthe HEPT class; Triangle/Abbott); CAPRAVIRINE™ (AG-1549/S-1153, a nextgeneration NNRTI with activity against viruses containing the K103Nmutation; Agouron); PNU-142721 (has 20-to 50-fold greater activity thanits predecessor delavirdine and is active against K103N mutants;Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivativesof efavirenz, designed to be active against viruses with the K103Nmutation; DuPont); GW-420867X (has 25-fold greater activity than HBY097and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A(naturally occurring agent from the latex tree; active against virusescontaining either or both the Y181C and K103N mutations); and Propolis(WO 99/49830).

Additional protease inhibitors include LOPINAVIR™ (ABT378/r; AbbottLaboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb);TIPRANAVIR™ (PNU-140690, a non-peptic dihydropyrone; Pharmacia &Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an indinaviranalog; Merck); DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776(a peptidomimetic with in vitro activity against proteaseinhibitor-resistant viruses; Agouron); VX-175/GW-433908 (phosphateprodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba); andAGENERASE™ (amprenavir; Glaxo Wellcome Inc.).

Additional antiretroviral agents include fusion inhibitors/gp41 binders.Fusion inhibitors/gp41 binders include T-20 (a peptide from residues643-678 of the HIV gp41 transmembrane protein ectodomain which binds togp41 in its resting state and prevents transformation to the fusogenicstate; Trimeris) and T-1249 (a second-generation fusion inhibitor;Trimeris).

Additional antiretroviral agents include fusion inhibitors/chemokinereceptor antagonists. Fusion inhibitors/chemokine receptor antagonistsinclude CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and itsanalogs, and ALX40-4C (a cationic peptide), T22 (an 18 amino acidpeptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonistssuch as RANTES (9-68), AOP-RANTES, NNY-RANTES, and TAK-779; andCCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Alsoincluded are CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetoragonists such as RANTES, SDF-1, MIP-1α, MIP-1β, etc., may also inhibitfusion.

Additional antiretroviral agents include integrase inhibitors. Integraseinhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (adicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and relatedanthraquinones; ZINTEVIR™ (AR 177, an oligonucleotide that probably actsat cell surface rather than being a true integrase inhibitor; Arondex);and naphthols such as those disclosed in WO 98/50347.

Additional antiretroviral agents include hydroxyurea-like compounds suchas BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst);ribonucleotide reductase inhibitors such as DIDOX™ (Molecules forHealth); inosine monophosphate dehydrogenase (IMPDH) inhibitors such asVX-497 (Vertex); and mycopholic acids such as CellCept (mycophenolatemofetil; Roche).

Additional antiretroviral agents include inhibitors of viral integrase,inhibitors of viral genome nuclear translocation such as arylenebis(methylketone) compounds; inhibitors of HIV entry such as AOP-RANTES,NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES andglycosaminoglycans (GAG), and AMD-3100; nucleocapsid zinc fingerinhibitors such as dithiane compounds; targets of HIV Tat and Rev; andpharmacoenhancers such as ABT-378.

Other antiretroviral therapies and adjunct therapies include cytokinesand lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKIN™(aldesleukin/L2-7001; Chiron), IL-4, IL-10, IL-12, and IL-13;interferons such as IFN-α2a; antagonists of TNFs, NFκB, GM-CSF, M-CSF,and IL-10; agents that modulate immune activation such as cyclosporinand prednisone; vaccines such as Remune™ (HIV Immunogen), APL 400-003(Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinantenvelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JR-FL protein, branched synthetic peptidederived from discontinuous gp120 C3/C4 domain, fusion-competentimmunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapiessuch as genetic suppressor elements (GSEs; WO 98/54366), and intrakines(genetically modified CC chemokines targetted to the ER to block surfaceexpression of newly synthesized CCR5 (Yang et al., PNAS 94:11567-72(1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as theanti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9,PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4,the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d,447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-αantibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptoragonists and antagonists such as TCDD, 3,3′,4,4′,5-pentachlorobiphenyl,3,3′,4,4′-tetrachlorobiphenyl, and α-naphthoflavone (WO 98/30213); andantioxidants such as γ-L-glutamyl-L-cysteine ethyl ester (γ-GCE; WO99/56764).

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In other embodiments, Therapeutics of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™,and/or AZITHROMYCIN™ to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with PYRIMETHAMINE™ and/orLEUCOVORIN™ to prophylactically treat or prevent an opportunisticToxoplasma gondii infection. In another specific embodiment,Therapeutics of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rapamycin, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.

In other embodiments, Therapeutics of the invention are administered incombination with immunosuppressive agents. Immunosuppressive agents thatmay be administered in combination with the Therapeutics of theinvention include, but are not limited to, steroids, cyclosporine,cyclosporine analogs, cyclophosphamide methylprednisone, prednisone,azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressiveagents that act by suppressing the function of responding T cells. Otherimmunosuppressive agents that may be administered in combination withthe Therapeutics of the invention include, but are not limited to,prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin,leflunomide, mizoribine (BREDININ™), brequinar, deoxyspergualin, andazaspirane (SKF 105685), ORTHOCLONE OKT® 3 (muromonab-CD3), SANDIMMUNE™,NEORAL™, SANGDYA™ (cyclosporine), PROGRAF® (FK506, tacrolimus),CELLCEPT® (mycophenolate motefil, of which the active metabolite ismycophenolic acid), IMURAN™ (azathioprine), glucocorticosteroids,adrenocortical steroids such as DELTASONE™ (prednisone) and HYDELTRASOL™(prednisolone), FOLEX™ and MEXATE™ (methotrxate), OXSORALEN-ULTRA™(methoxsalen) and RAPAMUNE™ (sirolimus). In a specific embodiment,immunosuppressants may be used to prevent rejection of organ or bonemarrow transplantation.

In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, ATGAM™(antithymocyte glubulin), and GAMIMUNE™. In a specific embodiment,Therapeutics of the invention are administered in combination withintravenous immune globulin preparations in transplantation therapy(e.g., bone marrow transplant).

In certain embodiments, the Therapeutics of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the Therapeuticsof the invention include, but are not limited to, corticosteroids (e.g.betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone,methylprednisolone, prednisolone, prednisone, and triamcinolone),nonsteroidal anti-inflammatory drugs (e.g., diclofenac, diflunisal,etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen,indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam,nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac,tenoxicam, tiaprofenic acid, and tolmetin.), as well as antihistamines,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-angiogenic agent.Anti-angiogenic agents that may be administered with the compositions ofthe invention include, but are not limited to, Angiostatin (Entremed,Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.),anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel(Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, TissueInhibitor of Metalloproteinase-2, VEGI, Plasminogen ActivatorInhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of thelighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono-and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude, but are not limited to, platelet factor 4; protamine sulphate;sulphated chitin derivatives (prepared from queen crab shells), (Murataet al., Cancer Res. 51:22-26, (1991)); Sulphated PolysaccharidePeptidoglycan Complex (SP-PG) (the function of this compound may beenhanced by the presence of steroids such as estrogen, and tamoxifencitrate); Staurosporine; modulators of matrix metabolism, including forexample, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline,Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, (1992)); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, (1992)); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,(1990)); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, (1987)); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4): 1659-1664, (1987)); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl4-chloroanthronilic acid disodium or “CCA”;(Takeuchi et al., Agents Actions 36:312-316, (1992)); andmetalloproteinase inhibitors such as BB94.

Additional anti-angiogenic factors that may also be utilized within thecontext of the present invention include Thalidomide, (Celgene, Warren,N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J. Folkman J Pediatr.Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C.Storgard et al., J Clin. Invest. 103:47-54 (1999));carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National CancerInstitute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE, Boston,Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.);TNP-470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca(London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251(PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin;Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide(Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat(AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex);Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and5-Fluorouracil.

Anti-angiogenic agents that may be administed in combination with thecompounds of the invention may work through a variety of mechanismsincluding, but not limited to, inhibiting proteolysis of theextracellular matrix, blocking the function of endothelialcell-extracellular matrix adhesion molecules, by antagonizing thefunction of angiogenesis inducers such as growth factors, and inhibitingintegrin receptors expressed on proliferating endothelial cells.Examples of anti-angiogenic inhibitors that interfere with extracellularmatrix proteolysis and which may be administered in combination with thecompositions of the invention include, but are not limited to, AG-3340(Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.),BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A(Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford,UK), and Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenicinhibitors that act by blocking the function of endothelialcell-extracellular matrix adhesion molecules and which may beadministered in combination with the compositions of the inventioninclude, but are not limited to, EMD-121974 (Merck KcgaA Darmstadt,Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg,Md.). Examples of anti-angiogenic agents that act by directlyantagonizing or inhibiting angiogenesis inducers and which may beadministered in combination with the compositons of the inventioninclude, but are not lmited to, Angiozyme (Ribozyme, Boulder, Colo.),Anti-VEGF antibody (Genentech, S. San Francisco, Calif.),PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. SanFrancisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, N.J.),and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectlyinhibit angiogenesis. Examples of indirect inhibitors of angiogenesiswhich may be administered in combination with the compositons of theinvention include, but are not limited to, IM-862 (Cytran, Kirkland,Wash.), Interferon-alpha, IL-12 (Roche, Nutley, N.J.), and Pentosanpolysulfate (Georgetown University, Washington, D.C.).

In particular embodiments, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of an autoimmune disease,such as for example, an autoimmune disease described herein.

In a particular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of arthritis. In a moreparticular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of rheumatoid arthritis.

In another embodiment, the polynucleotides encoding a polypeptide of thepresent invention are administered in combination with an angiogenicprotein, or polynucleotides encoding an angiogenic protein. Examples ofangiogenic proteins that may be administered with the compositions ofthe invention include, but are not limited to, acidic and basicfibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growthfactor alpha and beta, platelet-derived endothelial cell growth factor,platelet-derived growth factor, tumor necrosis factor alpha, hepatocytegrowth factor, insulin-like growth factor, colony stimulating factor,macrophage colony stimulating factor, granulocyte/macrophage colonystimulating factor, and nitric oxide synthase.

In additional embodiments, compositions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the Therapeuticsof the invention include, but are not limited to alkylating agents suchas nitrogen mustards (for example, Mechlorethamine, cyclophosphamide,Cyclophosphamide Ifosfamide, Melphalan (L-sarcolysin), andChlorambucil), ethylenimines and methylmelamines (for example,Hexamethylmelamine and Thiotepa), alkyl sulfonates (for example,Busulfan), nitrosoureas (for example, Carmustine (BCNU), Lomustine(CCNU), Semustine (methyl-CCNU), and Streptozocin (streptozotocin)),triazenes (for example, Dacarbazine (DTIC;dimethyltriazenoimidazolecarboxamide)), folic acid analogs (for example,Methotrexate (amethopterin)), pyrimidine analogs (for example,Fluorouacil (5-fluorouracil; 5-FU), Floxuridine (fluorodeoxyuridine;FudR), and Cytarabine (cytosine arabinoside)), purine analogs andrelated inhibitors (for example, Mercaptopurine (6-mercaptopurine;6-MP), Thioguanine (6-thioguanine; TG), and Pentostatin(2′-deoxycoformycin)), vinca alkaloids (for example, Vinblastine (VLB,vinblastine sulfate)) and Vincristine (vincristine sulfate)),epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics(for example, Dactinomycin (actinomycin D), Daunorubicin (daunomycin;rubidomycin), Doxorubicin, Bleomycin, Plicamycin (mithramycin), andMitomycin (mitomycin C), enzymes (for example, L-Asparaginase),biological response modifiers (for example, Interferon-alpha andinterferon-alpha-2b), platinum coordination compounds (for example,Cisplatin (cis-DDP) and Carboplatin), anthracenedione (Mitoxantrone),substituted ureas (for example, Hydroxyurea), methylhydrazinederivatives (for example, Procarbazine (N-methylhydrazine; MIH),adrenocorticosteroids (for example, Prednisone), progestins (forexample, Hydroxyprogesterone caproate, Medroxyprogesterone,Medroxyprogesterone acetate, and Megestrol acetate), estrogens (forexample, Diethylstilbestrol (DES), Diethylstilbestrol diphosphate,Estradiol, and Ethinyl estradiol), antiestrogens (for example,Tamoxifen), androgens (Testosterone proprionate, and Fluoxymesterone),antiandrogens (for example, Flutamide), gonadotropin-releasing horomoneanalogs (for example, Leuprolide), other hormones and hormone analogs(for example, methyltestosterone, estramustine, estramustine phosphatesodium, chlorotrianisene, and testolactone), and others (for example,dicarbazine, glutamic acid, and mitotane).

In one embodiment, the compositions of the invention are administered incombination with one or more of the following drugs: infliximab (alsoknown as Remicade™ Centocor, Inc.), Trocade (Roche, RO-32-3555),Leflunomide (also known as Arava™ from Hoechst Marion Roussel), Kineret™(an IL-1 Receptor antagonist also known as Anakinra from Amgen, Inc.)

In a specific embodiment, compositions of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or combination of one or more of the components of CHOP.In one embodiment, the compositions of the invention are administered incombination with anti-CD20 antibodies, human monoclonal anti-CD20antibodies. In another embodiment, the compositions of the invention areadministered in combination with anti-CD20 antibodies and CHOP, oranti-CD20 antibodies and any combination of one or more of thecomponents of CHOP, particularly cyclophosphamide and/or prednisone. Ina specific embodiment, compositions of the invention are administered incombination with Rituximab. In a further embodiment, compositions of theinvention are administered with Rituximab and CHOP, or Rituximab and anycombination of one or more of the components of CHOP, particularlycyclophosphamide and/or prednisone. In a specific embodiment,compositions of the invention are administered in combination withtositumomab. In a further embodiment, compositions of the invention areadministered with tositumomab and CHOP, or tositumomab and anycombination of one or more of the components of CHOP, particularlycyclophosphamide and/or prednisone. The anti-CD20 antibodies mayoptionally be associated with radioisotopes, toxins or cytotoxicprodrugs.

In another specific embodiment, the compositions of the invention areadministered in combination Zevalin™. In a further embodiment,compositions of the invention are administered with Zevalin™ and CHOP,or Zevalin™ and any combination of one or more of the components ofCHOP, particularly cyclophosphamide and/or prednisone. Zevalin™ may beassociated with one or more radisotopes. Particularly preferred isotopesare ⁹⁰Y and ¹¹¹In.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

In one embodiment, the Therapeutics of the invention are administered incombination with members of the TNF family. TNF, TNF-related or TNF-likemolecules that may be administered with the Therapeutics of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899),endokine-alpha (International Publication No. WO 98/07880), OPG, andneutrokine-alpha (International Publication No. WO 98/18921, OX40, andnerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3(International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TRANK, TR9 (International Publication No. WO 98/56892),TR10(International Publication No. WO 98/54202), 312C2 (InternationalPublication No. WO 98/06842), and TR12, and soluble forms CD154, CD70,and CD153.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are herein incorporated by reference in their entireties.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the Therapeutics of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to,granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostim,LEUKINE™, PROKINE™), granulocyte colony stimulating factor (G-CSF)(filgrastim, NEUPOGEN™), macrophage colony stimulating factor (M-CSF,CSF-1) erythropoietin (epoetin alfa, EPOGEN™, PROCRIT™), stem cellfactor (SCF, c-kit ligand, steel factor), megakaryocyte colonystimulating factor, PIXY321 (a GMCSF/IL-3 fusion protein), interleukins,especially any one or more of IL-1 through IL-12, interferon-gamma, orthrombopoietin.

In certain embodiments, Therapeutics of the present invention areadministered in combination with adrenergic blockers, such as, forexample, acebutolol, atenolol, betaxolol, bisoprolol, carteolol,labetalol, metoprolol, nadolol, oxprenolol, penbutolol, pindolol,propranolol, sotalol, and timolol.

In another embodiment, the Therapeutics of the invention areadministered in combination with an antiarrhythmic drug (e.g.,adenosine, amidoarone, bretylium, digitalis, digoxin, digitoxin,diliazem, disopyramide, esmolol, flecainide, lidocaine, mexiletine,moricizine, phenytoin, procainamide, N-acetyl procainamide, propafenone,propranolol, quinidine, sotalol, tocainide, and verapamil).

In another embodiment, the Therapeutics of the invention areadministered in combination with diuretic agents, such as carbonicanhydrase-inhibiting agents (e.g., acetazolamide, dichlorphenamide, andmethazolamide), osmotic diuretics (e.g., glycerin, isosorbide, mannitol,and urea), diuretics that inhibit Na⁺-K⁺-2Cl⁻ symport (e.g., furosemide,bumetanide, azosemide, piretanide, tripamide, ethacrynic acid,muzolimine, and torsemide), thiazide and thiazide-like diuretics (e.g.,bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide,hydroflumethiazide, methyclothiazide, polythiazide, trichormethiazide,chlorthalidone, indapamide, metolazone, and quinethazone), potassiumsparing diuretics (e.g., amiloride and triamterene), andmineralcorticoid receptor antagonists (e.g., spironolactone, canrenone,and potassium canrenoate).

In one embodiment, the Therapeutics of the invention are administered incombination with treatments for endocrine and/or hormone imbalancedisorders. Treatments for endocrine and/or hormone imbalance disordersinclude, but are not limited to, ¹²⁷I, radioactive isotopes of iodinesuch as ¹³¹I and ¹²³I; recombinant growth hormone, such as HUMATROPE™(recombinant somatropin); growth hormone analogs such as PROTROPIN™(somatrem); dopamine agonists such as PARLODEL™ (bromocriptine);somatostatin analogs such as SANDOSTATIN™ (octreotide); gonadotropinpreparations such as PREGNYL™, A.P.L.™ and PROFASI™ (chorionicgonadotropin (CG)), PERGONAL™ (menotropins), and METRODIN™(urofollitropin (uFSH)); synthetic human gonadotropin releasing hormonepreparations such as FACTREL™ and LUTREPULSE™ (gonadorelinhydrochloride); synthetic gonadotropin agonists such as LUPRON™(leuprolide acetate), SUPPRELIN™ (histrelin acetate), SYNAREL™(nafarelin acetate), and ZOLADEX™ (goserelin acetate); syntheticpreparations of thyrotropin-releasing hormone such as RELEFACT TRH™ andTHYPINONE™ (protirelin); recombinant human TSH such as THYROGEN™;synthetic preparations of the sodium salts of the natural isomers ofthyroid hormones such as L-T₄™, SYNTHROID™ and LEVOTHROID™(levothyroxine sodium), L-T₃™, CYTOMEL™ and TRIOSTAT™ (liothyroinesodium), and THYROLAR™ (liotrix); antithyroid compounds such as6-n-propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazoleand TAPAZOLE™ (methimazole), NEO-MERCAZOLE™ (carbimazole);beta-adrenergic receptor antagonists such as propranolol and esmolol;Ca²⁺ channel blockers; dexamethasone and iodinated radiological contrastagents such as TELEPAQUE™ (iopanoic acid) and ORAGRAFIN™ (sodiumipodate).

Additional treatments for endocrine and/or hormone imbalance disordersinclude, but are not limited to, estrogens or congugated estrogens suchas ESTRACE™ (estradiol), ESTINYL™ (ethinyl estradiol), PREMARIN™,ESTRATAB™, ORTHO-EST™, OGEN™ and estropipate (estrone), ESTROVIS™(quinestrol), ESTRADERM™ (estradiol), DELESTROGEN™ and VALERGEN™(estradiol valerate), DEPO-ESTRADIOL CYPIONATE™ and ESTROJECT LA™(estradiol cypionate); antiestrogens such as NOLVADEX™ (tamoxifen),SEROPHENE™ and CLOMID™ (clomiphene); progestins such as DURALUTIN™(hydroxyprogesterone caproate), MPA™ and DEPO-PROVERA™(medroxyprogesterone acetate), PROVERA™ and CYCRIN™ (MPA), MEGACE™(megestrol acetate), NORLUTIN™ (norethindrone), and NORLUTATE™ andAYGESTIN™ (norethindrone acetate); progesterone implants such asNORPLANT SYSTEM™ (subdermal implants of norgestrel); antiprogestins suchas RU⁴⁸⁶™ (mifepristone); hormonal contraceptives such as ENOVID™(norethynodrel plus mestranol), PROGESTASERT™ (intrauterine device thatreleases progesterone), LOESTRIN™, BREVICON™, MODICON™, GENORA™,NELONA™, NORINYL™, OVACON-35™ and OVACON-50™ (ethinylestradiol/norethindrone), LEVLEN™, NORDETTE™, TRI-LEVLEN™ andTRIPHASIL-21™ (ethinyl estradiol/levonorgestrel) LO/OVRAL™ and OVRAL™(ethinyl estradiol/norgestrel), DEMULEN™ (ethinyl estradiol/ethynodioldiacetate), NORINYL™, ORTHO-NOVUM™, NORETHIN™, GENORA™, and NELOVA™(norethindrone/mestranol), DESOGEN™ and ORTHO-CEPT™ (ethinylestradiol/desogestrel), ORTHO-CYCLEN™ and ORTHO-TRICYCLEN™ (ethinylestradiol/norgestimate), MICRONOR™ and NOR-QD™ (norethindrone), andOVRETTE™ (norgestrel).

Additional treatments for endocrine and/or hormone imbalance disordersinclude, but are not limited to, testosterone esters such as methenoloneacetate and testosterone undecanoate; parenteral and oral androgens suchas TESTOJECT-50™ (testosterone), TESTEX™ (testosterone propionate),DELATESTRYL™ (testosterone enanthate), DEPO-TESTOSTERONE™ (testosteronecypionate), DANOCRINE™ (danazol), HALOTESTIN™ (fluoxymesterone), ORETONMETHYL™, TESTRED™ and VIRILON™ (methyltestosterone), and OXANDRIN™(oxandrolone); testosterone transdermal systems such as TESTODERM™;androgen receptor antagonist and 5-alpha-reductase inhibitors such asANDROCUR™ (cyproterone acetate), EULEXIN™ (flutamide), and PROSCAR™(finasteride); adrenocorticotropic hormone preparations such asCORTROSYN™ (cosyntropin); adrenocortical steroids and their syntheticanalogs such as ACLOVATE™ (alclometasone dipropionate), CYCLOCORT™(amcinonide), BECLOVENT™ and VANCERIL™ (beclomethasone dipropionate),CELESTONE™ (betamethasone), BENISONE™ and UTICORT™ (betamethasonebenzoate), DIPROSONE™ (betamethasone dipropionate), CELESTONE PHOSPHATE™(betamethasone sodium phosphate), CELESTONE SOLUSPAN™ (betamethasonesodium phosphate and acetate), BETA-VAL™ and VALISONE™ (betamethasonevalerate), TEMOVATE™ (clobetasol propionate), CLODERM™ (clocortolonepivalate), CORTEF™ and HYDROCORTONE™ (cortisol (hydrocortisone)),HYDROCORTONE ACETATE™ (cortisol (hydrocortisone) acetate), LOCOID™(cortisol (hydrocortisone) butyrate), HYDROCORTONE PHOSPHATE™ (cortisol(hydrocortisone) sodium phosphate), A-HYDROCORT™ and SOLU CORTEF™(cortisol (hydrocortisone) sodium succinate), WESTCORT™ (cortisol(hydrocortisone) valerate), CORTISONE ACETATE™ (cortisone acetate),DESOWEN™ and TRIDESILON™ (desonide), TOPICORT™ (desoximetasone),DECADRON™ (dexamethasone), DECADRON LA™ (dexamethasone acetate),DECADRON PHOSPHATE™ and HEXADROL PHOSPHATE™ (dexamethasone sodiumphosphate), FLORONE™ and MAXIFLOR™ (diflorasone diacetate), FLORINEFACETATE™ (fludrocortisone acetate), AEROBID™ and NASALIDE™(flunisolide), FLUONID™ and SYNALAR™ (fluocinolone acetonide), LIDEX™(fluocinonide), FLUOR-OP™ and FML™ (fluorometholone), CORDRAN™(flurandrenolide), HALOG™ (halcinonide), HMS LIZUIFILM™ (medrysone),MEDROL™ (methylprednisolone), DEPO-MEDROL™ and MEDROL ACETATE™(methylprednisone acetate), A-METHAPRED™ and SOLUMEDROL™(methylprednisolone sodium succinate), ELOCON™ (mometasone furoate),HALDRONE™ (paramethasone acetate), DELTA-CORTEF™ (prednisolone),ECONOPRED™ (prednisolone acetate), HYDELTRASOL™ (prednisolone sodiumphosphate), HYDELTRA-T.B.A™ (prednisolone tebutate), DELTASONE™(prednisone), ARISTOCORT™ and KENACORT™ (triamcinolone), KENALOG™(triamcinolone acetonide), ARISTOCORT™ and KENACORT DIACETATE™(triamcinolone diacetate), and ARISTOSPAN™ (triamcinolone hexacetonide);inhibitors of biosynthesis and action of adrenocortical steroids such asCYTADREN™ (aminoglutethimide), NIZORAL™ (ketoconazole), MODRASTANE™(trilostane), and METOPIRONE™ (metyrapone).

Additional treatments for endocrine and/or hormone imbalance disordersinclude, but are not limited to bovine, porcine or human insulin ormixtures thereof; insulin analogs; recombinant human insulin such asHUMULIN™ and NOVOLIN™; oral hypoglycemic agents such as ORAMIDE™ andORINASE™ (tolbutamide), DIABINESE™ (chlorpropamide), TOLAMIDE™ andTOLINASE™ (tolazamide), DYMELOR™ (acetohexamide), glibenclamide,MICRONASE™, DIBETA™ and GLYNASE™ (glyburide), GLUCOTROL™ (glipizide),and DIAMICRON™ (gliclazide), GLUCOPHAGE™ (metformin), PRECOSE™(acarbose), AMARYL™ (glimepiride), and ciglitazone; thiazolidinediones(TZDs) such as rosiglitazone, AVANDIA™ (rosiglitazone maleate) ACTOS™(piogliatazone), and troglitazone; alpha-glucosidase inhibitors; bovineor porcine glucagon; somatostatins such as SANDOSTATIN™ (octreotide);and diazoxides such as PROGLYCEM™ (diazoxide). In still otherembodiments, Therapeutics of the invention are administered incombination with one or more of the following: a biguanide antidiabeticagent, a glitazone antidiabetic agent, and a sulfonylurea antidiabeticagent.

In one embodiment, the Therapeutics of the invention are administered incombination with treatments for uterine motility disorders. Treatmentsfor uterine motility disorders include, but are not limited to, estrogendrugs such as conjugated estrogens (e.g., PREMARIN® and ESTRATAB™),estradiols (e.g., CLIMARA® and ALORA®), estropipate, andchlorotrianisene; progestin drugs (e.g., AMEN® (medroxyprogesterone),MICRONOR® (norethidrone acetate), PROMETRIUM® progesterone, andmegestrol acetate); and estrogen/progesterone combination therapies suchas, for example, conjugated estrogens/medroxyprogesterone (e.g.,PREMPRO™ and PREMPHASE®) and norethindrone acetate/ethinyl estsradiol(e.g., FEMHRT™).

In an additional embodiment, the Therapeutics of the invention areadministered in combination with drugs effective in treating irondeficiency and hypochromic anemias, including but riot limited to,ferrous sulfate (iron sulfate, FEOSOL™), ferrous fumarate (e.g.,FEOSTAT™), ferrous gluconate (e.g., FERGON™), polysaccharide-ironcomplex (e.g., NIFEREX™), iron dextran injection (e.g., INFED™), cupricsulfate, pyroxidine, riboflavin, Vitamin B₁₂, cyancobalamin injection(e.g., REDISOL™, RUBRAMIN PC™), hydroxocobalamin, folic acid (e.g.,FOLVITE™), leucovorin (folinic acid, 5-CHOH4PteGlu, citrovorum factor)or WELLCOVORIN (Calcium salt of leucovorin), transferrin or ferritin.

In certain embodiments, the Therapeutics of the invention areadministered in combination with agents used to treat psychiatricdisorders. Psychiatric drugs that may be administered with theTherapeutics of the invention include, but are not limited to,antipsychotic agents (e.g., chlorpromazine, chlorprothixene, clozapine,fluphenazine, haloperidol, loxapine, mesoridazine, molindone,olanzapine, perphenazine, pimozide, quetiapine, risperidone,thioridazine, thiothixene, trifluoperazine, and triflupromazine),antimanic agents (e.g., carbamazepine, divalproex sodium, lithiumcarbonate, and lithium citrate), antidepressants (e.g., amitriptyline,amoxapine, bupropion, citalopram, clomipramine, desipramine, doxepin,fluvoxamine, fluoxetine, imipramine, isocarboxazid, maprotiline,mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine,protriptyline, sertraline, tranylcypromine, trazodone, trimipramine, andvenlafaxine), antianxiety agents (e.g., alprazolam, buspirone,chlordiazepoxide, clorazepate, diazepam, halazepam, lorazepam, oxazepam,and prazepam), and stimulants (e.g., d-amphetamine, methylphenidate, andpemoline).

In other embodiments, the Therapeutics of the invention are administeredin combination with agents used to treat neurological disorders.Neurological agents that may be administered with the Therapeutics ofthe invention include, but are not limited to, antiepileptic agents(e.g., carbamazepine, clonazepam, ethosuximide, phenobarbital,phenytoin, primidone, valproic acid, divalproex sodium, felbamate,gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine,topiramate, zonisamide, diazepam, lorazepam, and clonazepam),antiparkinsonian agents (e.g., levodopa/carbidopa, selegiline,amantidine, bromocriptine, pergolide, ropinirole, pramipexole,benztropine; biperiden; ethopropazine; procyclidine; trihexyphenidyl,tolcapone), and ALS therapeutics (e.g. riluzole).

In another embodiment, Therapeutics of the invention are administered incombination with vasodilating agents and/or calcium channel blockingagents. Vasodilating agents that may be administered with theTherapeutics of the invention include, but are not limited to,Angiotensin Converting Enzyme (ACE) inhibitors (e.g., papaverine,isoxsuprine, benazepril, captopril, cilazapril, enalapril, enalaprilat,fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril,spirapril, trandolapril, and nylidrin), and nitrates (e.g., isosorbidedinitrate, isosorbide mononitrate, and nitroglycerin). Examples ofcalcium channel blocking agents that may be administered in combinationwith the Therapeutics of the invention include, but are not limited toamlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine,nicardipine, nifedipine, nimodipine, and verapamil.

In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 24 Method of Treating Decreased Levels of the Polypeptide

The present invention relates to a method for treating an individual inneed of an increased level of a polypeptide of the invention in the bodycomprising administering to such an individual a composition comprisinga therapeutically effective amount of an agonist of the invention(including polypeptides of the invention). Moreover, it will beappreciated that conditions caused by a decrease in the standard ornormal expression level of a secreted protein in an individual can betreated by administering the polypeptide of the present invention,preferably in the secreted form. Thus, the invention also provides amethod of treatment of an individual in need of an increased level ofthe polypeptide comprising administering to such an individual aTherapeutic comprising an amount of the polypeptide to increase theactivity level of the polypeptide in such an individual.

For example, a patient with decreased levels of a polypeptide receives adaily dose 0.1-100 ug/kg of the polypeptide for six consecutive days.Preferably, the polypeptide is in the secreted form. The exact detailsof the dosing scheme, based on administration and formulation, areprovided in Example 23.

Example 25 Method of Treating Increased Levels of the Polypeptide

The present invention also relates to a method of treating an individualin need of a decreased level of a polypeptide of the invention in thebody comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an antagonist of theinvention (including polypeptides and antibodies of the invention).

In one, example, antisense technology is used to inhibit production of apolypeptide of the present invention. This technology is one example ofa method of decreasing levels of a polypeptide, preferably a secretedform, due to a variety of etiologies, such as cancer. For example, apatient diagnosed with abnormally increased levels of a polypeptide isadministered intravenously antisense polynucleotides at 0.5, 1.0, 1.5,2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a7-day rest period if the treatment was well tolerated. The formulationof the antisense polynucleotide is provided in Example 23.

Example 26 Method of Treatment Using Gene Therapy-Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing a polypeptide, onto a patient. Generally, fibroblasts areobtained from a subject by skin biopsy. The resulting tissue is placedin tissue-culture medium and separated into small pieces. Small chunksof the tissue are placed on a wet surface of a tissue culture flask,approximately ten pieces are placed in each flask. The flask is turnedupside down, closed tight and left at room temperature over night. After24 hours at room temperature, the flask is inverted and the chunks oftissue remain fixed to the bottom of the flask and fresh media (e.g.,Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added.The flasks are then incubated at 37 degree C. for approximately oneweek.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and Hindi HI and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 1 using primers andhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellstransduced with the vector. The packaging cells now produce infectiousviral particles containing the gene (the packaging cells are nowreferred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether protein isproduced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 27 Gene Therapy Using Endogenous Genes Corresponding ToPolynucleotides of the Invention

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous polynucleotide sequence ofthe invention with a promoter via homologous recombination as described,for example, in U.S. Pat. No.: 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/2941 1, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired.

Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous polynucleotide sequence, flanking the promoter. Thetargeting sequence will be sufficiently near the 5′ end of thepolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination. The promoter and thetargeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenouspolynucleotide sequence. This results in the expression ofpolynucleotide corresponding to the polynucleotide in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the locus corresponding to thepolynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst,N.Y.) is digested with HindIII. The CMV promoter is amplified by PCRwith an XbaI site on the 5′ end and a BamHI site on the 3′ end. Twonon-coding sequences are amplified via PCR: one non-coding sequence(fragment 1) is amplified with a HindIII site at the 5′ end and an Xbasite at the 3′ end; the other non-coding sequence (fragment 2) isamplified with a BamHI site at the 5′ end and a HindIII site at the 3′end. The CMV promoter and the fragments (1 and 2) are digested with theappropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI;fragment 2—BamHI) and ligated together. The resulting ligation productis digested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5.×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 28 Method of Treatment Using Gene Therapy-In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) sequences into an animal to increase or decreasethe expression of the polypeptide. The polynucleotide of the presentinvention may be operatively linked to a promoter or any other geneticelements necessary for the expression of the polypeptide by the targettissue. Such gene therapy and delivery techniques and methods are knownin the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos.5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res.35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997);Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., GeneTher. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290(1996) (incorporated herein by reference).

The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare 10 free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the polynucleotides of the present invention may alsobe delivered in liposome formulations (such as those taught in FelgnerP. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. etal. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods wellknown to those skilled in the art.

The polynucleotide vector constructs used in the gene therapy method arepreferably constructs that will not integrate into the host genome norwill they contain sequences that allow for replication. Any strongpromoter known to those skilled in the art can be used for driving theexpression of DNA. Unlike other gene therapies techniques, one majoradvantage of introducing naked nucleic acid sequences into target cellsis the transitory nature of the polynucleotide synthesis in the cells.Studies have shown that non-replicating DNA sequences can be introducedinto cells to provide production of the desired polypeptide for periodsof up to six months.

The polynucleotide construct can be delivered to the interstitial spaceof tissues within the an animal, including of muscle, skin, brain, lung,liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked polynucleotide injection, an effective dosage amount ofDNA or RNA will be in the range of from about 0.05 g/kg body weight toabout 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected polynucleotide in muscle in vivois determined as follows. Suitable template DNA for production of mRNAcoding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The template DNA is injected in 0.1 ml of carrier in a 1 ccsyringe through a 27 gauge needle over one minute, approximately 0.5 cmfrom the distal insertion site of the muscle into the knee and about 0.2cm deep. A suture is placed over the injection site for futurelocalization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 umcross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for protein expression maybe done in a similar fashion except that quadriceps from different miceare harvested at different times. Persistence of DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in humans andother animals using naked DNA.

Example 29 Transgenic Animals

The polypeptides of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of polypeptides of the present invention, studying diseases,disorders, and/or conditions associated with aberrant expression, and inscreening for compounds effective in ameliorating such diseases,disorders, and/or conditions.

Example 30 Knock-Out Animals

Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of polypeptides of the presentinvention, studying diseases, disorders, and/or conditions associatedwith aberrant expression, and in screening for compounds effective inameliorating such diseases, disorders, and/or conditions.

Example 31 Production of an Antibody

Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing polypeptide(s) of the invention areadministered to an animal to induce the production of sera containingpolyclonal antibodies. In a preferred method, a preparation ofpolypeptide(s) of the invention is prepared and purified to render itsubstantially free of natural contaminants. Such a preparation is thenintroduced into an animal in order to produce polyclonal antisera ofgreater specific activity.

Monoclonal antibodies specific for polypeptide(s) of the invention areprepared using hybridoma technology. (Kohler et al., Nature 256:495(1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al.,Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: MonoclonalAntibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)).In general, an animal (preferably a mouse) is immunized withpolypeptide(s) of the invention, or, more preferably, with a secretedpolypeptide-expressing cell. Such polypeptide-expressing cells arecultured in any suitable tissue culture medium, preferably in Earle'smodified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56° C.), and supplemented with about 10 g/l ofnonessential amino acids, about 1,000 U/ml of penicillin, and about 100μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP2O), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the polypeptide(s) of theinvention.

Alternatively, additional antibodies capable of binding polypeptide(s)of the invention can be produced in a two-step procedure usinganti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and therefore, it is possible toobtain an antibody which binds to a second antibody. In accordance withthis method, protein specific antibodies are used to immunize an animal,preferably a mouse. The splenocytes of such an animal are then used toproduce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to thepolypeptide(s) of the invention protein-specific antibody can be blockedby polypeptide(s) of the invention. Such antibodies compriseanti-idiotypic antibodies to the polypeptide(s) of the inventionprotein-specific antibody and are used to immunize an animal to induceformation of further polypeptide(s) of the invention protein-specificantibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric and humanized antibodies are known in theart and are discussed herein. (See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature314:268 (1985).)

Isolation Of Antibody Fragments Directed polypeptide(s) of the InventionFrom A Library Of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againstpolypeptide(s) of the invention to which the donor may or may not havebeen exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein byreference in its entirety).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 109 E. coli harboring thephagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 withshaking. Five ml of this culture is used to innoculate 50 ml of2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 ug/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing. These ELISApositive clones may also be further characterized by techniques known inthe art, such as, for example, epitope mapping, binding affinity,receptor signal transduction, ability to block or competitively inhibitantibody/antigen binding, and competitive agonistic or antagonisticactivity.

Example 32 Assays Detecting Stimulation or Inhibition of B cellProliferation and Differentiation

Generation of functional humoral immune responses requires both solubleand cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

One of the best studied classes of B-cell co-stimulatory proteins is theTNF-superfamily. Within this family CD40, CD27, and CD30 along withtheir respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

In Vitro Assay—Purified polypeptides of the invention, or truncatedforms thereof, is assessed for its ability to induce activation,proliferation, differentiation or inhibition and/or death in B-cellpopulations and their precursors. The activity of the polypeptides ofthe invention on purified human tonsillar B cells, measuredqualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessedin a standard B-lymphocyte co-stimulation assay in which purifiedtonsillar B cells are cultured in the presence of either formalin-fixedStaphylococcus aureus Cowan I (SAC) or immobilized anti-human IgMantibody as the priming agent. Second signals such as IL-2 and IL-15synergize with SAC and IgM crosslinking to elicit B cell proliferationas measured by tritiated-thymidine incorporation. Novel synergizingagents can be readily identified using this assay. The assay involvesisolating human tonsillar B cells by magnetic bead (MACS) depletion ofCD3-positive cells. The resulting cell population is greater than 95% Bcells as assessed by expression of CD45R(B220).

Various dilutions of each sample are placed into individual wells of a96-well plate to which are added 10⁵ B-cells suspended in culture medium(RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin, 10ug/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of 150ul. Proliferation or inhibition is quantitated by a 20 h pulse (1uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

In Vivo Assay—BALB/c mice are injected (i.p.) twice per day with bufferonly, or 2 mg/Kg of a polypeptide of the invention, or truncated formsthereof. Mice receive this treatment for 4 consecutive days, at whichtime they are sacrificed and various tissues and serum collected foranalyses. Comparison of H&E sections from normal spleens and spleenstreated with polypeptides of the invention identify the results of theactivity of the polypeptides on spleen cells, such as the diffusion ofperi-arterial lymphatic sheaths, and/or significant increases in thenucleated cellularity of the red pulp regions, which may indicate theactivation of the differentiation and proliferation of B-cellpopulations. Immunohistochemical studies using a B cell marker,anti-CD45R(B220), are used to determine whether any physiologicalchanges to splenic cells, such as splenic disorganization, are due toincreased B-cell representation within loosely defined B-cell zones thatinfiltrate established T-cell regions.

Flow cytometric analyses of the spleens from mice treated withpolypeptide is used to indicate whether the polypeptide specificallyincreases the proportion of ThB+, CD45R(B220)dull B cells over thatwhich is observed in control mice.

Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andpolypeptide-treated mice.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides of theinvention (e.g., gene therapy), agonists, and/or antagonists ofpolynucleotides or polypeptides of the invention.

Example 33 T Cell Proliferation Assay

Proliferation Assay for Resting PBLs.

A CD3-induced proliferation assay is performed on PBMCs and is measuredby the uptake of ³H-thymidine. The assay is performed as follows.Ninety-six well plates are coated with 100 microliters per well of mAbto CD3 (HIT₃a, Pharmingen) or isotype-matched control mAb (B33.1)overnight at 4° C. (1 microgram/ml in 0.05M bicarbonate buffer, pH 9.5),then washed three times with PBS. PBMC are isolated by F/H gradientcentrifugation from human peripheral blood and added to quadruplicatewells (5×10⁴/well) of mAb coated plates in RPMI containing 10% FCS andP/S in the presence of varying concentrations of TNF Delta and/or TNFEpsilon protein (total volume 200 microliters). Relevant protein bufferand medium alone are controls. After 48 hr. culture at 37° C., platesare spun for 2 min. at 1000 rpm and 100 microliters of supernatant isremoved and stored −20° C. for measurement of IL-2 (or other cytokines)if effect on proliferation is observed. Wells are supplemented with 100microliters of medium containing 0.5 microcuries of ³H-thymidine andcultured at 37° C. for 18-24 hr. Wells are harvested and incorporationof ³H-thymidine used as a measure of proliferation. Anti-CD3 alone isthe positive control for proliferation. IL-2 (100 U/ml) is also used asa control which enhances proliferation. Control antibody which does notinduce proliferation of T cells is used as the negative controls for theeffects of TNF Delta and/or TNF Epsilon proteins.

Alternatively, a proliferation assay on resting PBL (peripheral bloodlymphocytes) is measured by the up-take of ³H-thymidine. The assay isperformed as follows. PBMC are isolated by Ficoll (LSM, ICNBiotechnologies, Aurora, Ohio) gradient centrifugation from humanperipheral blood, and are cultured overnight in 10% (Fetal Calf Serum,Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). Thisovernight incubation period allows the adherent cells to attach to theplastic, which results in a lower background in the assay as there arefewer cells that can act as antigen presenting cells or that might beproducing growth factors. The following day the non-adherent cells arecollected, washed and used in the proliferation assay. The assay isperformed in a 96 well plate using 2×10⁴ cells/well in a final volume of200 microliters. The supernatants (e.g., CHO or 293T supernatants)expressing the protein of interest are tested at a 30% final dilution,therefore 60 ul are added to 140 ul of 10% FCS/RPMI containing thecells. Control supernatants are used at the same final dilution andexpress the following proteins: vector (negative control), IL-2 (*),IFN□, TNF□, IL-10 and TR2. In addition to the control supernatants,recombinant human IL-2 (R & D Systems, Minneapolis, Minn.) at a finalconcentration of 100 ng/ml is also used. After 24 hours of culture, eachwell is pulsed with 1 uCi of ³H-thymidine (Nen, Boston, Mass.). Cellsare then harvested 20 hours following pulsing and incorporation of³H-thymidine is used as a measure of proliferation. Results areexpressed as an average of triplicate samples plus or minus standarderror.

(*) The amount of the control cytokines IL-2, IFN□, TNF□ and IL-10produced in each transfection varies between 300 pg to 5 ng/ml.

Costimulation Assay.

A costimulation assay on resting PBL (peripheral blood lymphocytes) isperformed in the presence of immobilized antibodies to CD3 and CD28. Theuse of antibodies specific for the invariant regions of CD3 mimic theinduction of T cell activation that would occur through stimulation ofthe T cell receptor by an antigen. Cross-linking of the TCR (firstsignal) in the absence of a costimulatory signal (second signal) causesvery low induction of proliferation and will eventually result in astate of “anergy”, which is characterized by the absence of growth andinability to produce cytokines. The addition of a costimulatory signalsuch as an antibody to CD28, which mimics the action of thecostimulatory molecule. B7-1 expressed on activated APCs, results inenhancement of T cell responses including cell survival and productionof IL-2. Therefore this type of assay allows to detect both positive andnegative effects caused by addition of supernatants expressing theproteins of interest on T cell proliferation.

The assay is performed as follows. Ninety-six well plates are coatedwith 100 ng/ml anti-CD3 and 5 ug/ml anti-CD28 (Pharmingen, San Diego,Calif.) in a final volume of 100 ul and incubated overnight at 4C.Plates are washed twice with PBS before use. PBMC are isolated by Ficoll(LSM, ICN Biotechnologies, Aurora, Ohio) gradient centrifugation fromhuman peripheral blood, and are cultured overnight in 10% FCS(Fetal CalfSerum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.).This overnight incubation period allows the adherent cells to attach tothe plastic, which results in a lower background in the assay as thereare fewer cells that can act as antigen presenting cells or that mightbe producing growth factors. The following day the non adherent cellsare collected, washed and used in the proliferation assay. The assay isperformed in a 96 well plate using 2×10⁴ cells/well in a final volume of200 ul. The supernatants (e.g., CHO or 293T supernatants) expressing theprotein of interest are tested at a 30% final dilution, therefore 60 ulare added to 140 ul of 10% FCS/RPMI containing the cells. Controlsupernatants are used at the same final dilution and express thefollowing proteins: vector only (negative control), IL-2, TFN□, TNF□,IL-10 and TR2. In addition to the control supernatants recombinant humanIL-2 (R & D Systems, Minneapolis, Minn.) at a final concentration of 10ng/ml is also used. After 24 hours of culture, each well is pulsed with1 uCi of ³H-thymidine (Nen, Boston, Mass.). Cells are then harvested 20hours following pulsing and incorporation of ³H-thymidine is used as ameasure of proliferation. Results are expressed as an average oftriplicate samples plus or minus standard error.

Costimulation Assay: IFN γ and IL-2 ELISA.

The assay is performed as follows. Twenty-four well plates are coatedwith either 300 ng/ml or 600 ng/ml anti-CD3 and 5 ug/ml anti-CD28(Pharmingen, San Diego, Calif.) in a final volume of 500 ul andincubated overnight at 4C. Plates are washed twice with PBS before use.PBMC are isolated by Ficoll (LSM, ICN Biotechnologies, Aurora, Ohio)gradient centrifugation from human peripheral blood, and are culturedovernight in 10% FCS(Fetal Calf Serum, Blofluids, Rockville. Md.)/RPMI(Gibco BRL, Gaithersburg, Md.). This overnight incubation period allowsthe adherent cells to attach to the plastic, which results in a lowerbackground in the assay as there are fewer cells that can act as antigenpresenting cells or that might be producing growth factors. Thefollowing day the non adherent cells are collected, washed and used inthe costimulation assay. The assay is performed in the pre-coatedtwenty-four well plate using 1×10⁵ cells/well in a final volume of 900ul. The supernatants (293T supernatants) expressing the protein ofinterest are tested at a 30% final dilution, therefore 300 ul are addedto 600 ul of 10% FCS/RPMI containing the cells. Control supernatants areused at the same final dilution and express the following proteins:vector only(negative control), IL-2, IFN□, IL-12 and IL-18. In additionto the control supernatants recombinant human IL-2 (all cytokines werepurchased from R & D Systems, Minneapolis, Minn.) at a finalconcentration of 10 ng/ml, IL-12 at a final concentration of 1 ng/ml andIL-18 at a final concentration of 50 ng/ml are also used. Controls andunknown samples are tested in duplicate. Supernatant samples (250 ul)are collected 2 days and 5 days after the beginning of the assay. ELISAsto test for IFN□ and IL-2 secretion are performed using kits purchasedfrom R & D Systems, (Minneapolis, Minn.). Results are expressed as anaverage of duplicate samples plus or minus standard error.

Proliferation Assay for Preactivated-Resting T cells.

A proliferation assay on preactivated-resting T cells is performed oncells that are previously activated with the lectin phytohemagglutinin(PHA). Lectins are polymeric plant proteins that can bind to residues onT cell surface glycoproteins including the TCR and act as polyclonalactivators. PBLs treated with PHA and then cultured in the presence oflow doses of IL-2 resemble effector T cells. These cells are generallymore sensitive to further activation induced by growth factors such asIL-2. This is due to the expression of high affinity IL-2 receptors thatallows this population to respond to amounts of IL-2 that are 100 foldlower than what would have an effect on a naïve T cell. Therefore theuse of this type of cells might enable to detect the effect of very lowdoses of an unknown growth factor, that would not be sufficient toinduce proliferation on resting (naïve) T cells.

The assay is performed as follows. PBMC are isolated by F/H gradientcentrifugation from human peripheral blood, and are cultured in 10%FCS(Fetal Calf Serum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL,Gaithersburg, Md.) in the presence of 2 ug/ml PHA (Sigma, Saint Louis,Mo.) for three days. The cells are then washed in PBS and cultured in10% FCS/RPMI in the presence of 5 ng/ml of human recombinant IL-2 (R & DSystems, Minneapolis, Minn.) for 3 days. The cells are washed and restedin starvation medium (1%FCS/RPMI) for 16 hours prior to the beginning ofthe proliferation assay. An aliquot of the cells is analyzed by FACS todetermine the percentage of T cells (CD3 positive cells) present; thisusually ranges between 93-97% depending on the donor. The assay isperformed in a 96 well plate using 2×10⁴ cells/well in a final volume of200 ul. The supernatants (e.g., CHO or 293T supernatants) expressing theprotein of interest are tested at a 30% final dilution, therefore 60 ulare added to 140 ul of in 10% FCS/RPMI containing the cells. Controlsupernatants are used at the same final dilution and express thefollowing proteins: vector (negative control), IL-2, IFN□, TNF□, IL-10and TR2. In addition to the control supernatants recombinant human IL-2at a final concentration of 10 ng/ml is also used. After 24 hours ofculture, each well is pulsed with 1 uCi of ³H-thymidine(Nen, Boston,Mass.). Cells are then harvested 20 hours following pulsing andincorporation of ³H-thymidine is used as a measure of proliferation.Results are expressed as an average of triplicate samples plus or minusstandard error.

The studies described in this example test activity of polypeptides ofthe invention. However, one skilled in the art could easily modify theexemplified studies to test the activity of polynucleotides of theinvention (e.g., gene therapy), agonists, and/or antagonists ofpolynucleotides or polypeptides of the invention.

Example 34 Effect of Polypeptides of the Invention on the Expression ofMHC Class II, Costimulatory and Adhesion Molecules and CellDifferentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CS F (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-α,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFCγRII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

FACS analysis of surface antigens is performed as follows. Cells aretreated 1-3 days with increasing concentrations of polypeptides of theinvention or LPS (positive control), washed with PBS containing 1% BSAand 0.02 miM sodium azide, and then incubated with 1:20 dilution ofappropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at4 degrees C. After an additional wash, the labeled cells are analyzed byflow cytometry on a FACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendriticcells, in particular IL-12, are important in the initiation of T-celldependent immune responses. IL-12 strongly influences the development ofTh1 helper T-cell immune response, and induces cytotoxic T and NK cellfunction. An ELISA is used to measure the IL-12 release as follows.Dendritic cells (10⁶/ml) are treated with increasing concentrations ofpolypeptides of the invention for 24 hours. LPS (100 ng/ml) is added tothe cell culture as positive control. Supernatants from the cellcultures are then collected and analyzed for IL-12 content usingcommercial ELISA kit (e.g, R & D Systems (Minneapolis, Minn.)). Thestandard protocols provided with the kits are used.

Effect on the expression of MHC Class II, costimulatory and adhesionmolecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations ofpolypeptides of the invention or LPS (positive control), washed with PBScontaining 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30minutes at 4 degrees C. After an additional wash, the labeled cells areanalyzed by flow cytometry on a FACScan (Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules thatactivate (or alternatively, inactivate) monocytes and/or increasemonocyte survival (or alternatively, decrease monocyte survival) areknown in the art and may routinely be applied to determine whether amolecule of the invention functions as an inhibitor or activator ofmonocytes. Polypeptides, agonists, or antagonists of the invention canbe screened using the three assays described below. For each of theseassays, Peripheral blood mononuclear cells (PBMC) are purified fromsingle donor leukopacks (American Red Cross, Baltimore, Md.) bycentrifugation through a Histopaque gradient (Sigma). Monocytes areisolated from PBMC by counterflow centrifugal elutriation.

Monocyte Survival Assay. Human peripheral blood monocytes progressivelylose viability when cultured in absence of serum or other stimuli. Theirdeath results from internally regulated process (apoptosis). Addition tothe culture of activating factors, such as TNF-alpha dramaticallyimproves cell survival and prevents DNA fragmentation. Propidium iodide(PI) staining is used to measure apoptosis as follows. Monocytes arecultured for 48 hours in polypropylene tubes in serum-free medium(positive control), in the presence of 100 ng/ml TNF-alpha (negativecontrol), and in the presence of varying concentrations of the compoundto be tested. Cells are suspended at a concentration of 2×10⁶/ml in PBScontaining PI at a final concentration of 5 μg/ml, and then incubaed atroom temperature for 5 minutes before FACScan analysis. PI uptake hasbeen demonstrated to correlate with DNA fragmentation in thisexperimental paradigm.

Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×10⁵ cells/mlwith increasing concentrations of the a polypeptide of the invention andunder the same conditions, but in the absence of the polypeptide. ForIL-12 production, the cells are primed overnight with IFN (100 U/ml) inpresence of a polypeptide of the invention. LPS (10 ng/ml) is thenadded. Conditioned media are collected after 24 h and kept frozen untiluse. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performedusing a commercially available ELISA kit (e.g, R & D Systems(Minneapolis, Minn.)) and applying the standard protocols provided withthe kit.

Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×10⁵cell/well. Increasing concentrations of polypeptides of the inventionare added to the wells in a total volume of 0.2 ml culture medium (RPMI1640+10% FCS, glutamine and antibiotics). After 3 days incubation, theplates are centrifuged and the medium is removed from the wells. To themacrophage monolayers, 0.2 ml per well of phenol red solution (140 mMNaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mMphenol red and 19 U/ml of HRPO) is added, together with the stimulant(200 nM PMA). The plates are incubated at 37° C. for 2 hours and thereaction is stopped by adding 20 μl IN NaOH per well. The absorbance isread at 610 nm. To calculate the amount of H₂O₂ produced by themacrophages, a standard curve of a H₂O₂ solution of known molarity isperformed for each experiment.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polypeptides,polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 35 Biological Effects of Polypeptides of the Invention

Astrocyte and Neuronal Assays:

Recombinant polypeptides of the invention, expressed in Escherichia coliand purified as described above, can be tested for activity in promotingthe survival, neurite outgrowth, or phenotypic differentiation ofcortical neuronal cells and for inducing the proliferation of glialfibrillary acidic protein immunopositive cells, astrocytes. Theselection of cortical cells for the bioassay is based on the prevalentexpression of FGF-1 and FGF-2 in cortical structures and on thepreviously reported enhancement of cortical neuronal survival resultingfrom FGF-2 treatment. A thymidine incorporation assay, for example, canbe used to elucidate a polypeptide of the invention's activity on thesecells.

Moreover, previous reports describing the biological effects of FGF-2(basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of a polypeptideof the invention to induce neurite outgrowth can be compared to theresponse achieved with FGF-2 using, for example, a thymidineincorporation assay.

Fibroblast and Endothelial Cell Assays

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.)and maintained in growth media from Clonetics. Dermal microvascularendothelial cells are obtained from Cell Applications (San Diego,Calif.). For proliferation assays, the human lung fibroblasts and dermal-microvascular endothelial cells can be cultured at 5,000 cells/well ina 96-well plate for one day in growth medium. The cells are thenincubated for one day in 0.1% BSA basal medium. After replacing themedium with fresh 0.1% BSA medium, the cells are incubated with the testproteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento,Calif.) is added to each well to a final concentration of 10%. The cellsare incubated for 4 hr. Cell viability is measured by reading in aCytoFluor fluorescence reader. For the PGE₂ assays, the human lungfibroblasts are cultured at 5,000 cells/well in a 96-well plate for oneday. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or polypeptides of the invention with or withoutIL-1α for 24 hours. The supernatants are collected and assayed for PGE₂by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the humanlung fibroblasts are cultured at 5,000 cells/well in a 96-well plate forone day. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or with or without polypeptides of the inventionIL-1α for 24 hours. The supernatants are collected and assayed for IL-6by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or polypeptides of theinvention for 3 days in basal medium before the addition of Alamar Blueto assess effects on growth of the fibroblasts. FGF-2 should show astimulation at 10-2500 ng/ml which can be used to compare stimulationwith polypeptides of the invention.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to adeficiency of striatal dopamine resulting from the degeneration of thenigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP⁺) and released.Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP⁺ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basicFGF) has trophic activity towards nigral dopaminergic neurons (Ferrariet al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, polypeptides of the invention can beevaluated to determine whether it has an action similar to that of FGF-2in enhancing dopaminergic neuronal survival in vitro and it can also betested in vivo for protection of dopaminergic neurons in the striatumfrom the damage associated with MPTP treatment. The potential effect ofa polypeptide of the invention is first examined in vitro in adopaminergic neuronal cell culture paradigm. The cultures are preparedby dissecting the midbrain floor plate from gestation day 14 Wistar ratembryos. The tissue is dissociated with trypsin and seeded at a densityof 200,000 cells/cm² on polyorthinine-laminin coated glass coverslips.The cells are maintained in Dulbecco's Modified Eagle's medium and F12medium containing hormonal supplements (N1). The cultures are fixed withparaformaldehyde after 8 days in vitro and are processed for tyrosinehydroxylase, a specific marker for dopminergic neurons,immunohistochemical staining. Dissociated cell cultures are preparedfrom embryonic rats. The culture medium is changed every third day andthe factors are also added at that time.

Since the dopaminergic neurons are isolated from animals at gestationday 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if a polypeptide of the invention acts to prolong thesurvival of dopaminergic neurons, it would suggest that the polypeptidemay be involved in Parkinson's Disease.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 36 The Effect of Polypeptides of the Invention on the Growth ofVascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at2-5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumis replaced with M199 containing 10% FBS, 8 units/ml heparin. Apolypeptide having the amino acid sequence of SEQ ID NO:Y, and positivecontrols, such as VEGF and basic FGF (bFGF) are added, at varyingconcentrations. On days 4 and 6, the medium is replaced. On day 8, cellnumber is determined with a Coulter Counter.

An increase in the number of HUVEC cells indicates that the polypeptideof the invention may proliferate vascular endothelial cells.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 37 Stimulatory Effect of Polypeptides of the Invention on theProliferation of Vascular Endothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetricMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 mL serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF₁₆₅ or a polypeptide of the invention in 0.5%FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours.20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed toincubate for 1 hour at 37° C. before measuring the absorbance at 490 nmin an ELISA plate reader. Background absorbance from control wells (somemedia, no cells) is subtracted, and seven wells are performed inparallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol.30A:512-518 (1994).

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 38 Inhibition of PDGF-Induced Vascular Smooth Muscle CellProliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrdincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then,the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h,immunocytochemistry is performed by using BrdUrd Staining Kit (ZymedLaboratories). In brief, the cells are incubated with the biotinylatedmouse anti-BrdUrd antibody at 4 degrees C. for 2 h after being exposedto denaturing solution and then incubated with thestreptavidin-peroxidase and diaminobenzidine. After counterstaining withhematoxylin, the cells are mounted for microscopic examination, and theBrdUrd-positive cells are counted. The BrdUrd index is calculated as apercent of the BrdUrd-positive cells to the total cell number. Inaddition, the simultaneous detection of the BrdUrd staining (nucleus)and the FITC uptake (cytoplasm) is performed for individual cells by theconcomitant use of bright field illumination and dark field-UVfluorescent illumination. See, Hayashida et al., J. Biol. Chem.6:271(36):21985-21992 (1996).

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 39 Stimulation of Endothelial Migration

This example will be used to explore the possibility that a polypeptideof the invention may stimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, Md.; Falk, W., etal., J. Immunological Methods 1980;33:239-247).Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um(Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for atleast 6 hours at room temperature and dried under sterile air. Testsubstances are diluted to appropriate concentrations in M199supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of thefinal dilution is placed in the lower chamber of the modified Boydenapparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures arewashed and trypsinized for the minimum time required to achieve celldetachment. After placing the filter between lower and upper chamber,2.5×10⁵ cells suspended in 50 ul M199 containing 1% FBS are seeded inthe upper compartment. The apparatus is then incubated for 5 hours at37° C. in a humidified chamber with 5% CO2 to allow cell migration.After the incubation period, the filter is removed and the upper side ofthe filter with the non-migrated cells is scraped with a rubberpoliceman. The filters are fixed with methanol and stained with a Giemsasolution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration isquantified by counting cells of three random high-power fields (40×) ineach well, and all groups are performed in quadruplicate.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 40 Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be amediator of vascular endothelium relaxation. Thus, activity of apolypeptide of the invention can be assayed by determining nitric oxideproduction by endothelial cells in response to the polypeptide.

Nitric oxide is measured in 96-well plates of confluent microvascularendothelial cells after 24 hours starvation and a subsequent 4 hrexposure to various levels of a positive control (such as VEGF-1) andthe polypeptide of the invention. Nitric oxide in the medium isdetermined by use of the Griess reagent to measure total nitrite afterreduction of nitric oxide-derived nitrate by nitrate reductase. Theeffect of the polypeptide of the invention on nitric oxide release isexamined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with aNO-specific polarographic electrode connected to a NO meter (Iso-NO,World Precision Instruments Inc.) (1049). Calibration of the NO elementsis performed according to the following equation:2KNO₂+2KI+2H₂SO₄6 2NO+I₂+2H₂O+2 K₂SO₄

The standard calibration curve is obtained by adding gradedconcentrations of KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing KI and H₂SO₄. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas (1050). The culture medium is removed and HUVECsare washed twice with Dulbecco's phosphate buffered saline. The cellsare then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates are kept on a slide warmer (Lab LineInstruments Inc.) To maintain the temperature at 37° C. The NO sensorprobe is inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) is used as apositive control. The amount of released NO is expressed as picomolesper 1×10⁶ endothelial cells. All values reported are means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

The studies described in this example tested activity of polypeptides ofthe invention. However, one skilled in the art could easily modify theexemplified studies to test the activity of polynucleotides (e.g., genetherapy), agonists, and/or antagonists of the invention.

Example 41 Effect of Polypepides of the Invention on Cord Formation inAngiogenesis

Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications' CADMEC Growth Medium and used at passage 5. Forthe in vitro angiogenesis assay, the wells of a 48-well cell cultureplate are coated with Cell Applications' Attachment Factor Medium (200ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wellsat 7,500 cells/well and cultured overnight in Growth Medium. The GrowthMedium is then replaced with 300 mg Cell Applications' Chord FormationMedium containing control buffer or a polypeptide of the invention (0.1to 100 ng/ml) and the cells are cultured for an additional 48 hr. Thenumbers and lengths of the capillary-like chords are quantitated throughuse of the Boeckeler VIA-170 video image analyzer. All assays are donein triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.b-esteradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 42 Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system toexamine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of polypeptides of the invention tostimulate angiogenesis in CAM can be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and theJapanese qual (Coturnix coturnix) are incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old qualembryos is studied with the following methods.

On Day 4 of development, a window is made into the egg shell of chickeggs. The embryos are checked for normal development and the eggs sealedwith cellotape. They are further incubated until Day 13. Thermanoxcoverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm indiameter. Sterile and salt-free growth factors are dissolved indistilled water and about 3.3 mg/5 ml are pipetted on the disks. Afterair-drying, the inverted disks are applied on CAM. After 3 days, thespecimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsedin 0.12 M sodium cacodylate buffer. They are photographed with a stereomicroscope [Wild M8] and embedded for semi-and ultrathin sectioning asdescribed above. Controls are performed with carrier disks alone.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 43 Angiogenesis Assay Using a Matrigel Implant in Mouse

In vivo angiogenesis assay of a polypeptide of the invention measuresthe ability of an existing capillary network to form new vessels in animplanted capsule of murine extracellular matrix material (Matrigel).The protein is mixed with the liquid Matrigel at 4 degree C. and themixture is then injected subcutaneously in mice where it solidifies.After 7 days, the solid “plug” of Matrigel is removed and examined forthe presence of new blood vessels. Matrigel is purchased from BectonDickinson Labware/Collaborative Biomedical Products.

When thawed at 4 degree C. the Matrigel material is a liquid. TheMatrigel is mixed with a polypeptide of the invention at 150 ng/ml at 4degrees C. and drawn into cold 3 ml syringes. Female C57B1/6 miceapproximately 8 weeks old are injected with the mixture of Matrigel andexperimental protein at 2 sites at the midventral aspect of the abdomen(0.5 ml/site). After 7 days, the mice are sacrificed by cervicaldislocation, the Matrigel plugs are removed and cleaned (i.e., allclinging membranes and fibrous tissue is removed). Replicate whole plugsare fixed in neutral buffered 10% formaldehyde, embedded in paraffin andused to produce sections for histological examination after stainingwith Masson's Trichrome. Cross sections from 3 different regions of eachplug are processed. Selected sections are stained for the presence ofvWF. The positive control for this assay is bovine basic FGF (150ng/ml). Matrigel alone is used to determine basal levels ofangiogenesis.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 44 Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of polynucleotides and polypeptides of theinvention on ischemia, a rabbit hindlimb ischemia model is created bysurgical removal of one femoral arteries as described previously(Takeshita et al., Am J. Pathol 147:1649-1660 (1995)). The excision ofthe femoral artery results in retrograde propagation of thrombus andocclusion of the external iliac artery. Consequently, blood flow to theischemic limb is dependent upon collateral vessels originating from theinternal iliac artery (Takeshita et al. Am J. Pathol 147:1649-1660(1995)). An interval of 10 days is allowed for post-operative recoveryof rabbits and development of endogenous collateral vessels. At 10 daypost-operatively (day 0), after performing a baseline angiogram, theinternal iliac artery of the ischemic limb is transfected with 500 mgnaked expression plasmid containing a polynucleotide of the invention byarterial gene transfer technology using a hydrogel-coated ballooncatheter as described (Riessen et al. Hum Gene Ther. 4:749-758 (1993);Leclerc et al. J. Clin. Invest. 90: 936-944 (1992)). When a polypeptideof the invention is used in the treatment, a single bolus of 500 mgpolypeptide of the invention or control is delivered into the internaliliac artery of the ischemic limb over a period of 1 min. through aninfusion catheter. On day 30, various parameters are measured in theserabbits: (a) BP ratio—The blood pressure ratio of systolic pressure ofthe ischemic limb to that of normal limb; (b) Blood Flow and FlowReserve—Resting FL: the blood flow during undilated condition and MaxFL: the blood flow during fully dilated condition (also an indirectmeasure of the blood vessel amount) and Flow Reserve is reflected by theratio of max FL: resting FL; (c) Angiographic Score—This is measured bythe angiogram of collateral vessels. A score is determined by thepercentage of circles in an overlaying grid that with crossing opacifiedarteries divided by the total number m the rabbit thigh; (d) Capillarydensity—The number of collateral capillaries determined in lightmicroscopic sections taken from hindlimbs.

The studies described in this example tested activity of polynucleotidesand polypeptides of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the agonists, and/orantagonists of the invention.

Example 45 Effect of Polypeptides of the Invention on Vasodilation

Since dilation of vascular endothelium is important in reducing bloodpressure, the ability of polypeptides of the invention to affect theblood pressure in spontaneously hypertensive rats (SHR) is examined.Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of thepolypeptides of the invention are administered to 13-14 week oldspontaneously hypertensive rats (SHR). Data are expressed as the mean+/−SEM. Statistical analysis are performed with a paired t-test andstatistical significance is defined as p<0.05 vs. the response to bufferalone.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 46 Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skin temperature, andfactor VIII immunohistochemistry or endothelial alkaline phosphatasereaction. Expression of polypeptides of the invention, during the skinischemia, is studied using in situ hybridization.

The study in this model is divided into three parts as follows:

Ischemic skin

Ischemic skin wounds

Normal wounds

The experimental protocol includes:

Raising a 3×4 cm, single pedicle full-thickness random skin flap(myocutaneous flap over the lower back of the animal).

An excisional wounding (4-6 mm in diameter) in the ischemic skin(skin-flap).

Topical treatment with a polypeptide of the invention of the excisionalwounds (day 0, 1, 2, 3, 4 post-wounding) at the following various dosageranges: 1 mg to 100 mg.

Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21 post-woundingfor histological, immunohistochemical, and in situ studies.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 47 Peripheral Arterial Disease Model

Angiogenic therapy using a polypeptide of the invention is a noveltherapeutic strategy to obtain restoration of blood flow around theischemia in case of peripheral arterial diseases. The experimentalprotocol includes:

One side of the femoral artery is ligated to create ischemic muscle ofthe hindlimb, the other side of hindlimb serves as a control.

A polypeptide of the invention, in a dosage range of 20 mg-500 mg, isdelivered intravenously and/or intramuscularly 3 times (perhaps more)per week for 2-3 weeks.

The ischemic muscle tissue is collected after ligation of the femoralartery at 1, 2, and 3 weeks for the analysis of expression of apolypeptide of the invention and histology. Biopsy is also performed onthe other side of normal muscle of the contralateral hindlimb.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 48 Ischemic Myocardial Disease Model

A polypeptide of the invention is evaluated as a potent mitogen capableof stimulating the development of collateral vessels, and restructuringnew vessels after coronary artery occlusion. Alteration of expression ofthe polypeptide is investigated in situ. The experimental protocolincludes:

The heart is exposed through a left-side thoracotomy in the rat.Immediately, the left coronary artery is occluded with a thin suture(6-0) and the thorax is closed.

A polypeptide of the invention, in a dosage range of 20 mg-500 mg, isdelivered intravenously and/or intramuscularly 3 times (perhaps more)per week for 2-4 weeks.

Thirty days after the surgery, the heart is removed and cross-sectionedfor morphometric and in situ analyzes.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 49 Rat Corneal Wound Healing Model

This animal model shows the effect of a polypeptide of the invention onneovascularization. The experimental protocol includes:

Making a 1-1.5 mm long incision from the center of cornea into thestromal layer. Inserting a spatula below the lip of the incision facingthe outer corner of the eye. Making a pocket (its base is 1-1.5 mm formthe edge of the eye). Positioning a pellet, containing 50 ng-5 ug of apolypeptide of the invention, within the pocket.

Treatment with a polypeptide of the invention can also be appliedtopically to the corneal wounds in a dosage range of 20 mg-500 mg (dailytreatment for five days).

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 50 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

Diabetic db+/db+ Mouse Model.

To demonstrate that a polypeptide of the invention accelerates thehealing process, the genetically diabetic mouse model of wound healingis used. The full thickness wound healing model in the db+/db+ mouse isa well characterized, clinically relevant and reproducible model ofimpaired wound healing. Healing of the diabetic wound is dependent onformation of granulation tissue and re-epithelialization rather thancontraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992);Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979);Coleman, D. L., Diabetes 31 (Suppl): 1-6 (1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J. Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

A polypeptide of the invention is administered using at a rangedifferent doses, from 4 mg to 500 mg per wound per day for 8 days invehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3)treated group.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64 mm², the corresponding size of the dermalpunch. Calculations are made using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with a polypeptide of the invention. This assessment includedverification of the presence of cell accumulation, inflammatory cells,capillaries, fibroblasts, re-epithelialization and epidermal maturity(Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibratedlens micrometer is used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA), in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer can serve as a positive tissuecontrol and human brain tissue can be used as a negative tissue control.Each specimen includes a section with omission of the primary antibodyand substitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, Glucocorticoids and Woundhealing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahlet al., J. Immunol. 115: 476-481 (1975);Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retardwound healing by inhibiting angiogenesis, decreasing vascularpermeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)),fibroblast proliferation, and collagen synthesis (Beck et al., GrowthFactors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797(1978)) and producing a transient reduction of circulating monocytes(Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl,“Glucocorticoids and wound healing”, In: Antiinflammatory SteroidAction: Basic and Clinical Aspects, Academic Press, New York, pp.280-302 (1989)). The systemic administration of steroids to impairedwound healing is a well establish phenomenon in rats (Beck et al.,Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61:703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In:Antiinflammatory Steroid Action: Basic and Clinical Aspects, AcademicPress, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad.Sci. USA 86: 2229-2233 (1989)).

To demonstrate that a polypeptide of the invention can accelerate thehealing process, the effects of multiple topical applications of thepolypeptide on full thickness excisional skin wounds in rats in whichhealing has been impaired by the systemic administration ofmethylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On theday of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1-5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson caliper. Woundsare considered healed if granulation tissue is no longer visible and thewound is covered by a continuous epithelium.

The polypeptide of the invention is administered using at a rangedifferent doses, from 4 mg to 500 mg per wound per day for 8 days invehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Four groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) treated groups.

Wound closure is analyzed by measuring the area in-the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1is 64 mm², the corresponding size of the dermal punch. Calculations aremade using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing an Olympus microtome. Routine hematoxylin-eosin (H&E) staining isperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin is improved by treatmentwith a polypeptide of the invention. A calibrated lens micrometer isused by a blinded observer to determine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 51 Lymphadema Animal Model

The purpose of this experimental approach is to create an appropriateand consistent lymphedema model for testing the therapeutic effects of apolypeptide of the invention in lymphangiogenesis and re-establishmentof the lymphatic circulatory system in the rat hind limb. Effectivenessis measured by swelling volume of the affected limb, quantification ofthe amount of lymphatic vasculature, total blood plasma protein, andhistopathology. Acute lymphedema is observed for 7-10 days. Perhaps moreimportantly, the chronic progress of the edema is followed for up to 3-4weeks.

Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately ˜350 g aredosed with Pentobarbital. Subsequently, the right legs are shaved fromknee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH.Blood is drawn for serum total protein testing. Circumference andvolumetric measurements are made prior to injecting dye into paws aftermarking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsalpaw). The intradermal dorsum of both right and left paws are injectedwith 0.05 ml of 1% Evan's Blue. Circumference and volumetricmeasurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is madecircumferentially allowing the femoral vessels to be located. Forcepsand hemostats are used to dissect and separate the skin flaps. Afterlocating the femoral vessels, the lymphatic vessel that runs along sideand underneath the vessel(s) is located. The main lymphatic vessels inthis area are then electrically coagulated suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosisand adductors) are bluntly dissected. The popliteal lymph node is thenlocated. The 2 proximal and 2 distal lymphatic vessels and distal bloodsupply of the popliteal node are then and ligated by suturing. Thepopliteal lymph node, and any accompanying adipose tissue, is thenremoved by cutting connective tissues.

Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (AJ Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of ˜0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw before operation and daily for 7 days are measured. The effectplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limbmovement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople then those 2 readings are averaged. Readings are taken from bothcontrol and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetizedwith Pentobarbital and are tested prior to surgery. For dailyvolumetrics animals are under brief halothane anesthetic (rapidimmobilization and quick recovery), both legs are shaved and equallymarked using waterproof marker on legs. Legs are first dipped in water,then dipped into instrument to each marked level then measured by Buxcoedema software(Chen/Victor). Data is recorded by one person, while theother is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serumseparated prior to surgery and then at conclusion for total protein andCa2+ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared fortissue collection. The limbs are amputated using a quillitine, then bothexperimental and control legs are cut at the ligature and weighed. Asecond weighing is done as the tibio-cacaneal joint is disarticulatedand the foot is weighed.

Histological Preparations: The transverse muscle located behind the knee(popliteal) area is dissected and arranged in a metal mold, filled withfreezeGel, dipped into cold methylbutane, placed into labeled samplebags at −80EC until sectioning. Upon sectioning, the muscle is observedunder fluorescent microscopy for lymphatics.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 52 Suppression of TNF Alpha-Induced Adhesion Molecule Expressionby a Polypeptide of the Invention

The recruitment of lymphocytes to areas of inflammation and angiogenesisinvolves specific receptor-ligand interactions between cell surfaceadhesion molecules (CAMs) on lymphocytes and the vascular endothelium.The adhesion process, in both normal and pathological settings, followsa multi-step cascade that involves intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelialleukocyte adhesion molecule-1 (E-selectin) expression on endothelialcells (EC). The expression of these molecules and others on the vascularendothelium determines the efficiency with which leukocytes may adhereto the local vasculature and extravasate into the local tissue duringthe development of an inflammatory response. The local concentration ofcytokines and growth factor participate in the modulation of theexpression of these CAMs.

Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine,is a stimulator of all three CAMs on endothelial cells and may beinvolved in a wide variety of inflammatory responses, often resulting ina pathological outcome.

The potential of a polypeptide of the invention to mediate a suppressionof TNF-a induced CAM expression can be examined. A modified ELISA assaywhich uses ECs as a solid phase absorbent is employed to measure theamount of CAM expression on TNF-a treated ECs when co-stimulated with amember of the FGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC)cultures are obtained from pooled cord harvests and maintained in growthmedium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCSand 1% penicillin/streptomycin in a 37 degree C. humidified incubatorcontaining 5% CO₂. HUVECs are seeded in 96-well plates at concentrationsof 1×10⁴ cells/well in EGM medium at 37 degree C. for 18-24 hrs or untilconfluent. The monolayers are subsequently washed 3 times with aserum-free solution of RPMI-1640 supplemented with 100 U/ml penicillinand 100 mg/ml streptomycin, and treated with a given cytokine and/orgrowth factor(s) for 24 h at 37 degree C. Following incubation, thecells are then evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard96 well plate to confluence. Growth medium is removed from the cells andreplaced with 90 ul of 199 Medium (10% FBS). Samples for testing andpositive or negative controls are added to the plate in triplicate (in10 ul volumes). Plates are incubated at 37 degree C. for either 5 h(selectin and integrin expression) or 24 h (integrin expression only).Plates are aspirated to remove medium and 100 μl of 0.1%paraformaldehyde-PBS(with Ca++ and Mg++) is added to each well. Platesare held at 4° C. for 30 min.

Fixative is then removed from the wells and wells are washed 1× withPBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA.

Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000dilution) to each well and incubated at 37° C. for 30 min. Wells arewashed X3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol PhosphatepNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPPsubstrate in glycine buffer is added to each test well. Standard wellsin triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000(10⁰)>10^(−0.5)>10⁻¹>10^(−1.5) 0.5 μl of each dilution is added totriplicate wells and the resulting AP content in each well is 5.50 ng,1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added toeach of the standard wells. The plate must be incubated at 37° C. for 4h. A volume of 50 μl of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results areindicated as amount of bound AP-conjugate in each sample.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 53 Assay for the Stimulation of Bone Marrow CD34+ CellProliferation

This assay is based on the ability of human CD34+ to proliferate in thepresence of hematopoietic growth factors and evaluates the ability ofisolated polypeptides expressed in mammalian cells to stimulateproliferation of CD34+ cells.

It has been previously shown that most mature precursors will respond toonly a single signal. More immature precursors require at least twosignals to respond. Therefore, to test the effect of polypeptides onhematopoietic activity of a wide range of progenitor cells, the assaycontains a given polypeptide in the presence or absence of otherhematopoietic growth factors. Isolated cells are cultured for 5 days inthe presence of Stem Cell Factor (SCF) in combination with testedsample. SCF alone has a very limited effect on the proliferation of bonemarrow (BM) cells, acting in such conditions only as a “survival”factor. However, combined with any factor exhibiting stimulatory effecton these cells (e.g., IL-3), SCF will cause a synergistic effect.Therefore, if the tested polypeptide has a stimulatory effect on ahematopoietic progenitors, such activity can be easily detected. Sincenormal BM cells have a low level of cycling cells, it is likely that anyinhibitory effect of a given polypeptide, or agonists or antagoniststhereof, might not be detected. Accordingly, assays for an inhibitoryeffect on progenitors is preferably tested in cells that are firstsubjected to in vitro stimulation with SCF+IL+3, and then contacted withthe compound that is being evaluated for inhibition of such inducedproliferation.

Briefly, CD34+ cells are isolated using methods known in the art. Thecells are thawed and resuspended in medium (QBSF 60 serum-free mediumwith 1% L-glutamine (500 ml) Quality Biological, Inc., Gaithersburg, Md.Cat#160-204-101). After several gentle centrifugation steps at 200×g,cells are allowed to rest for one hour. The cell count is adjusted to2.5×10⁵ cells/ml. During this time, 100 μl of sterile water is added tothe peripheral wells of a 96-well plate. The cytokines that can betested with a given polypeptide in this assay is rhSCF (R&D Systems,Minneapolis, Minn., Cat#255-SC) at 50 ng/ml alone and in combinationwith rhSCF and rhIL-3 (R&D Systems, Minneapolis, Minn., Cat#203-ML) at30 ng/ml. After one hour, 10 μl of prepared cytokines, 50 μl SID(supernatants at 1:2 dilution=50 μl) and 20 μl of diluted cells areadded to the media which is already present in the wells to allow for afinal total volume of 100 μl. The plates are then placed in a 37° C./15%CO₂ incubator for five days.

Eighteen hours before the assay is harvested, 0.5 μCi/well of [3H]Thymidine is added in a 10 μl volume to each well to determine theproliferation rate. The experiment is terminated by harvesting the cellsfrom each 96-well plate to a filtermat using the Tomtec Harvester 96.After harvesting, the filtermats are dried, trimmed and placed intoOmniFilter assemblies consisting of one OmniFilter plate and oneOmniFilter Tray. 60 μl Microscint is added to each well and the platesealed with TopSeal-A press-on sealing film A bar code 15 sticker isaffixed to the first plate for counting. The sealed plates is thenloaded and the level of radioactivity determined via the Packard TopCount and the printed data collected for analysis. The level ofradioactivity reflects the amount of cell proliferation.

The studies described in this example test the activity of a givenpolypeptide to stimulate bone marrow CD34+ cell proliferation. Oneskilled in the art could easily modify the exemplified studies to testthe activity of polynucleotides (e.g., gene therapy), antibodies,agonists, and/or antagonists and fragments and variants thereof. As anonlimiting example, potential antagonists tested in this assay would beexpected to inhibit cell proliferation in the presence of cytokinesand/or to increase the inhibition of cell proliferation in the presenceof cytokines and a given polypeptide. In contrast, potential agoniststested in this assay would be expected to enhance cell proliferationand/or to decrease the inhibition of cell proliferation in the presenceof cytokines and a given polypeptide.

The ability of a gene to stimulate the proliferation of bone marrowCD34+ cells indicates that polynucleotides and polypeptidescorresponding to the gene are useful for the diagnosis and treatment ofdisorders affecting the immune system and hematopoiesis. Representativeuses are described in the “Immune Activity” and “Infectious Disease”sections above, and elsewhere herein.

Example 54 Assay for Extracellular Matrix Enhanced Cell Response (EMECR)

The objective of the Extracellular Matrix Enhanced Cell Response (EMECR)assay is to identify gene products (e.g., isolated polypeptides) thatact on the hematopoietic stem cells in the context of the extracellularmatrix (ECM) induced signal.

Cells respond to the regulatory factors in the context of signal(s)received from the surrounding microenvironment. For example,fibroblasts, and endothelial and epithelial stem cells fail to replicatein the absence of signals from the ECM. Hematopoietic stem cells canundergo self-renewal in the bone marrow, but not in in vitro suspensionculture. The ability of stem cells to undergo self-renewal in vitro isdependent upon their interaction with the stromal cells and the ECMprotein fibronectin (fn). Adhesion of cells to fn is mediated by theα₅.β₁ and α₄.β₁ integrin receptors, which are expressed by human andmouse hematopoietic stem cells. The factor(s) which integrate with theECM environment and responsible for stimulating stem cell self-renewalhas not yet been identified. Discovery of such factors should be ofgreat interest in gene therapy and bone marrow transplant applications

Briefly, polystyrene, non tissue culture treated, 96-well plates arecoated with fn fragment at a coating concentration of 0.2 μg/cm². Mousebone marrow cells are plated (1,000 cells/well ) in 0.2 ml of serum-freemedium. Cells cultured in the presence of IL-3 (5 ng/ml )+SCF (50 ng/ml)would serve as the positive control, conditions under which littleself-renewal but pronounced differentiation of the stem cells is to beexpected. Gene products are tested with appropriate negative controls inthe presence and absence of SCF(5.0 ng/ml), where test factor supernatesrepresent 10% of the total assay volume. The plated cells are thenallowed to grow by incubating in a low oxygen environment (5% CO₂, 7%O₂, and 88% N₂ ) tissue culture incubator for 7 days. The number ofproliferating cells within the wells is then quantitated by measuringthymidine incorporation into cellular DNA. Verification of the positivehits in the assay will require phenotypic characterization of the cells,which can be accomplished by scaling up of the culture system and usingappropriate antibody reagents against cell surface antigens and FACScan.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides (e.g., gene therapy), antibodies,agonists, and/or antagonists and fragments and variants thereof.

If a particular gene product is found to be a stimulator ofhematopoietic progenitors, polynucleotides and polypeptidescorresponding to the gene may be useful for the diagnosis and treatmentof disorders affecting the immune system and hematopoiesis.Representative uses are described in the “Immune Activity” and“Infectious Disease” sections above, and elsewhere herein. The geneproduct may also be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types.

Additionally, the polynucleotides and/or polypeptides of the gene ofinterest and/or agonists and/or antagonists thereof, may also beemployed to inhibit the proliferation and differentiation ofhematopoietic cells and therefore may be employed to protect bone marrowstem cells from chemotherapeutic agents during chemotherapy. Thisantiproliferative effect may allow administration of higher doses ofchemotherapeutic agents and, therefore, more effective chemotherapeutictreatment.

Moreover, polynucleotides and polypeptides corresponding to the gene ofinterest may also be useful for the treatment and diagnosis ofhematopoietic related disorders such as, for example, anemia,pancytopenia, leukopenia, thrombocytopenia or leukemia since stromalcells are important in the production of cells of hematopoieticlineages. The uses include bone marrow cell ex-vivo culture, bone marrowtransplantation, bone marrow reconstitution, radiotherapy orchemotherapy of neoplasia.

Example 55 Human Dermal Fibroblast and Aortic Smooth Muscle CellProliferation

The polypeptide of interest is added to cultures of normal human dermalfibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC) and twoco-assays are performed with each sample. The first assay examines theeffect of the polypeptide of interest on the proliferation of normalhuman dermal fibroblasts (NHDF) or aortic smooth muscle cells (AoSMC).Aberrant growth of fibroblasts or smooth muscle cells is a part ofseveral pathological processes, including fibrosis, and restenosis. Thesecond assay examines IL6 production by both NHDF and SMC. IL6production is an indication of functional activation. Activated cellswill have increased production of a number of cytokines and otherfactors, which can result in a proinflammatory or immunomodulatoryoutcome. Assays are run with and without co-TNFa stimulation, in orderto check for costimulatory or inhibitory activity.

Briefly, on day 1, 96-well black plates are set up with 1000 cells/well(NHDF) or 2000 cells/well (AoSMC) in 100 μl culture media. NHDF culturemedia contains: Clonetics FB basal media, 1 mg/ml hFGF, 5 mg/ml insulin,50 mg/ml gentamycin, 2% FBS, while AoSMC culture media containsClonetics SM basal media, 0.5 μg/ml hEGF, 5 mg/ml insulin, 1 μg/ml hFGF,50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 5% FBS. After incubation @37° C. for at least 4-5 hours culture media is aspirated and replacedwith growth arrest media. Growth arrest media for NHDF containsfibroblast basal media, 50 mg/ml gentamycin, 2% FBS, while growth arrestmedia for AoSMC contains SM basal media, 50 mg/ml gentamycin, 50 μg/mlAmphotericin B, 0.4% FBS. Incubate at 37C until day 2.

On day 2, serial dilutions and templates of the polypeptide of interestare designed which should always include media controls andknown-protein controls. For both stimulation and inhibition experiments,proteins are diluted in growth arrest media. For inhibition experiments,TNFa is added to a final concentration of 2 ng/ml (NHDF) or 5 ng/ml(AoSMC). Then add ⅓ vol media containing controls or supernatants andincubate at 37C/5% CO₂ until day 5.

Transfer 60 μl from each well to another labeled 96-well plate, coverwith a plate-sealer, and store at 4C until Day 6 (for IL6 ELISA). To theremaining 100 μl in the cell culture plate, aseptically add Alamar Bluein an amount equal to 10% of the culture volume (10 μl). Return platesto incubator for 3 to 4 hours. Then measure fluorescence with excitationat 530 nm and emission at 590 nm using the CytoFluor. This yields thegrowth stimulation/inhibition data.

On day 5, the IL6 ELISA is performed by coating a 96 well plate with50-100 ul/well of Anti-Human IL6 Monoclonal antibody diluted in PBS, pH7.4, incubate ON at room temperature.

On day 6, empty the plates into the sink and blot on paper towels.Prepare Assay Buffer containing PBS with 4% BSA. Block the plates with200 μl/well of Pierce Super Block blocking buffer in PBS for 1-2 hr andthen wash plates with wash buffer (PBS, 0.05% Tween-20). Blot plates onpaper towels. Then add 50 μl/well of diluted Anti-Human IL-6 Monoclonal,Biotin-labeled antibody at 0.50 mg/ml. Make dilutions of IL-6 stock inmedia (30, 10, 3, 1, 0.3, 0 ng/ml). Add duplicate samples to top row ofplate. Cover the plates and incubate for 2 hours at RT on shaker.

Wash plates with wash buffer and blot on paper towels. Dilute EU-labeledStreptavidin 1:1000 in Assay buffer, and add 100 μl/well. Cover theplate and incubate 1 h at RT. Wash plates with wash buffer. Blot onpaper towels.

Add 100 μl/well of Enhancement Solution. Shake for 5 minutes. Read theplate on the Wallac DELFIA Fluorometer. Readings from triplicate samplesin each assay were tabulated and averaged.

A positive result in this assay suggests AoSMC cell proliferation andthat the gene product of interest may be involved in dermal fibroblastproliferation and/or smooth muscle cell proliferation. A positive resultalso suggests many potential uses of polypeptides, polynucleotides,agonists and/or antagonists of the gene/gene product of interest. Forexample, inflammation and immune responses, wound healing, andangiogenesis, as detailed throughout this specification. Particularly,polypeptides of the gene product and polynucleotides of the gene may beused in wound healing and dermal regeneration, as well as the promotionof vasculargenesis, both of the blood vessels and lymphatics. The growthof vessels can be used in the treatment of, for example, cardiovasculardiseases. Additionally, antagonists of polypeptides of the gene productand polynucleotides of the gene may be useful in treating diseases,disorders, and/or conditions which involve angiogenesis by acting as ananti-vascular (e.g., anti-angiogenesis). These diseases, disorders,and/or conditions are known in the art and/or are described herein, suchas, for example, malignancies, solid tumors, benign tumors, for examplehemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenicgranulomas; artheroscleric plaques; ocular angiogenic diseases, forexample, diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma, retrolentalfibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormalblood vessel growth) of the eye; rheumatoid arthritis; psoriasis;delayed wound healing; endometriosis; vasculogenesis; granulations;hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma;vascular adhesions; myocardial angiogenesis; coronary collaterals;cerebral collaterals; arteriovenous malformations; ischemic limbangiogenesis; Osler-Webber Syndrome; plaque neovascularization;telangiectasia; hemophiliac joints; angiofibroma; fibromusculardysplasia; wound granulation; Crohn's disease; and atherosclerosis.Moreover, antagonists of polypeptides of the gene product andpolynucleotides of the gene may be useful in treatinganti-hyperproliferative diseases and/or anti-inflammatory known in theart and/or described herein.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides (e.g., gene therapy), antibodies,agonists, and/or antagonists and fragments and variants thereof.

Example 56 Cellular Adhesion Molecule (CAM) Expression on EndothelialCells

The recruitment of lymphocytes to areas of inflammation and angiogenesisinvolves specific receptor-ligand interactions between cell surfaceadhesion molecules (CAMs) on lymphocytes and the vascular endothelium.The adhesion process, in both normal and pathological settings, followsa multi-step cascade that involves intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelialleukocyte adhesion molecule-1 (E-selectin) expression on endothelialcells (EC). The expression of these molecules and others on the vascularendothelium determines the efficiency with which leukocytes may adhereto the local vasculature and extravasate into the local tissue duringthe development of an inflammatory response. The local concentration ofcytokines and growth factor participate in the modulation of theexpression of these CAMs.

Briefly, endothelial cells (e.g., Human Umbilical Vein Endothelial cells(HUVECs)) are grown in a standard 96 well plate to confluence, growthmedium is removed from the cells and replaced with 100 μl of 199 Medium(10% fetal bovine serum (FBS)). Samples for testing and positive ornegative controls are added to the plate in triplicate (in 10 μlvolumes). Plates are then incubated at 37° C. for either 5 h (selectinand integrin expression) or 24 h (integrin expression only). Plates areaspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS(withCa++ and Mg++) is added to each well. Plates are held at 4° C. for 30min. Fixative is removed from the wells and wells are washed 1× withPBS(+Ca,Mg)+0.5% BSA and drained. 10 μl of diluted primary antibody isadded to the test and control wells. Anti-ICAM-1-Biotin,Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at aconcentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody).Cells are incubated at 37° C. for 30 min. in a humidified environment.Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA. 20 μl of dilutedExtrAvidin-Alkaline Phosphotase (1:5,000 dilution, refered to herein asthe working dilution) are added to each well and incubated at 37° C. for30 min. Wells are washed three times with PBS(+Ca,Mg)+0.5% BSA. Dissolve1 tablet of p-Nitrophenol Phosphate pNPP per 5 ml of glycine buffer (pH10.4). 100 μl of pNPP substrate in glycine buffer is added to each testwell. Standard wells in triplicate are prepared from the workingdilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer:1:5,000 (10⁰)>10^(−0.5)>10⁻¹>10^(−1.5) 0.5 μl of each dilution is addedto triplicate wells and the resulting AP content in each well is 5.50ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent is then added toeach of the standard wells. The plate is incubated at 37° C. for 4 h. Avolume of 50 μl of 3M NaOH is added to all wells. The plate is read on aplate reader at 405 nm using the background subtraction option on blankwells filled with glycine buffer only. Additionally, the template is setup to indicate the concentration of AP-conjugate in each standard well[5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount ofbound AP-conjugate in each sample.

Example 57 Alamar Blue Endothelial Cells Proliferation Assay

This assay may be used to quantitatively determine protein mediatedinhibition of bFGF-induced proliferation of Bovine Lymphatic EndothelialCells (LECs), Bovine Aortic Endothelial Cells (BAECs) or HumanMicrovascular Uterine Myometrial Cells (UTMECs). This assay incorporatesa fluorometric growth indicator based on detection of metabolicactivity. A standard Alamar Blue Proliferation Assay is prepared inEGM-2MV with 10 ng/ml of bFGF added as a source of endothelial cellstimulation. This assay may be used with a variety of endothelial cellswith slight changes in growth medium and cell concentration. Dilutionsof the protein batches to be tested are diluted as appropriate.Serum-free medium (GIBCO SFM) without bFGF is used as a non-stimulatedcontrol and Angiostatin or TSP-1 are included as a known inhibitorycontrols.

Briefly, LEC, BAECs or UTMECs are seeded in growth media at a density of5000 to 2000 cells/well in a 96 well plate and placed at 37-C overnight.After the overnight incubation of the cells, the growth media is removedand replaced with GIBCO EC-SFM. The cells are treated with theappropriate dilutions of the protein of interest or control proteinsample(s) (prepared in SFM) in triplicate wells with additional bFGF toa concentration of 10 ng/ml. Once the cells have been treated with thesamples, the plate(s) is/are placed back in the 37° C. incubator forthree days. After three days 10 ml of stock alamar blue (BiosourceCat#DAL1100) is added to each well and the plate(s) is/are placed backin the 37° C. incubator for four hours. The plate(s) are then read at530 nm excitation and 590 nm emission using the CytoFluor fluorescencereader. Direct output is recorded in relative fluorescence units.

Alamar blue is an oxidation-reduction indicator that both fluoresces andchanges color in response to chemical reduction of growth mediumresulting from cell growth. As cells grow in culture, innate metabolicactivity results in a chemical reduction of the immediate surroundingenvironment. Reduction related to growth causes the indicator to changefrom oxidized (non-fluorescent blue) form to reduced (fluorescent red)form. i.e. stimulated proliferation will produce a stronger signal andinhibited proliferation will produce a weaker signal and the totalsignal is proportional to the total number of cells as well as theirmetabolic activity. The background level of activity is observed withthe starvation medium alone. This is compared to the output observedfrom the positive control samples (bFGF in growth medium) and proteindilutions.

Example 58 Detection of Inhibition of a Mixed Lymphocyte Reaction

This assay can be used to detect and evaluate inhibition of a MixedLymphocyte Reaction (MLR) by gene products (e.g., isolatedpolypeptides). Inhibition of a MLR may be due to a direct effect on cellproliferation and viability, modulation of costimulatory molecules oninteracting cells, modulation of adhesiveness between lymphocytes andaccessory cells, or modulation of cytokine production by accessorycells. Multiple cells may be targeted by these polypeptides since theperipheral blood mononuclear fraction used in this assay includes T, Band natural killer lymphocytes, as well as monocytes and dendriticcells.

Polypeptides of interest found to inhibit the MLR may find applicationin diseases associated with lymphocyte and monocyte activation orproliferation. These include, but are not limited to, diseases such asasthma, arthritis, diabetes, inflammatory skin conditions, psoriasis,eczema, systemic lupus erythematosus, multiple sclerosis,glomerulonephritis, inflammatory bowel disease, crohn's disease,ulcerative colitis, arteriosclerosis, cirrhosis, graft vs. host disease,host vs. graft disease, hepatitis, leukemia and lymphoma.

Briefly, PBMCs from human donors are purified by density gradientcentrifugation using Lymphocyte Separation Medium (LSM®, density 1.0770g/ml, Organon Teknika Corporation, West Chester, Pa.). PBMCs from twodonors are adjusted to 2×10⁶ cells/ml in RPMI-1640 (Life Technologies,Grand Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCsfrom a third donor is adjusted to 2×10⁵ cells/ml. Fifty microliters ofPBMCs from each donor is added to wells of a 96-well round bottommicrotiter plate. Dilutions of test materials (50 μl) is added intriplicate to microtiter wells. Test samples (of the protein ofinterest) are added for final dilution of 1:4; rhuIL-2 (R&D Systems,Minneapolis, Minn., catalog number 202-IL) is added to a finalconcentration of 1 μg/ml; anti-CD4 mAb (R&D Systems, clone 34930.11,catalog number MAB379) is added to a final concentration of 10 μg/ml.Cells are cultured for 7-8 days at 37° C. in 5% CO₂, and 1 μC of [³H]thymidine is added to wells for the last 16 hrs of culture. Cells areharvested and thymidine incorporation determined using a PackardTopCount. Data is expressed as the mean and standard deviation oftriplicate determinations.

Samples of the protein of interest are screened in separate experimentsand compared to the negative control treatment, anti-CD4 mAb, whichinhibits proliferation of lymphocytes and the positive controltreatment, IL-2 (either as recombinant material or supernatant), whichenhances proliferation of lymphocytes.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides (e.g., gene therapy), antibodies,agonists, and/or antagonists and fragments and variants thereof.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims. The entire disclosure of each documentcited (including patents, patent applications, journal articles,abstracts, laboratory manuals, books, or other disclosures) in theBackground of the Invention, Detailed Description, and Examples ishereby incorporated herein by reference. Further, the hard copy of thesequence listing submitted herewith and the corresponding computerreadable form are both incorporated herein by reference in theirentireties. Additionally, the contents of International Application No.PCT/US98/21142 and of Provisional Applications Ser. Nos. 60/061,463,60/061,529, 60/071,498, 60/061,527, 60/061,536 and 60/061,532 are allhereby incorporated by reference in their entirety.

TABLE 6 Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met1 A . . B . . . −0.93 0.37 * * . −0.30 0.53 Lys 2 A . . B . . . −0.540.63 * * −0.60 0.34 Phe 3 A . . B . . . −1.04 0.20 * * . −0.30 0.45 Leu4 A . . B . . . −1.47 0.46 * * . −0.60 0.32 Leu 5 A . . B . . . −1.890.53 * * . −0.60 0.13 Asp 6 A . . B . . . −2.10 1.21 * * . −0.60 0.12Ile 7 A . . B . . . −2.96 1.11 * * . −0.60 0.12 Leu 8 A . . B . . .−2.47 1.11 . * . −0.60 0.12 Leu 9 A . . B . . . −2.47 0.86 . . . −0.600.11 Leu 10 A . . B . . . −2.47 1.54 . * . −0.60 0.14 Leu 11 A . . B . .. −3.36 1.54 . . . −0.60 0.14 Pro 12 A . . B . . . −3.32 1.54 . . .−0.60 0.11 Leu 13 A . . B . . . −3.18 1.50 . . . −0.60 0.10 Leu 14 . . BB . . . −2.67 1.39 . . . −0.60 0.07 Ile 15 . . B B . . . −2.67 1.09 . .. −0.60 0.06 Val 16 . . B B . . . −1.86 1.34 . . . −0.60 0.06 Cys 17 . .B B . . . −1.94 0.66 . . . −0.60 0.12 Ser 18 A . . B . . . −1.83 0.36 *. . −0.30 0.23 Leu 19 A A . . . . . −1.88 0.46 * * . −0.60 0.27 Glu 20 AA . B . . . −0.94 0.46 * * . −0.60 0.38 Ser 21 A A . B . . . −0.90−0.11 * . . 0.30 0.56 Phe 22 A A . B . . . −0.93 0.19 * * . −0.30 0.56Val 23 A A . B . . . −1.52 0.29 * * . −0.30 0.28 Lys 24 A A . B . . .−0.92 0.97 * * . −0.60 0.15 Leu 25 A A . B . . . −0.88 1.01 . * . −0.260.26 Phe 26 A A . B . . . −0.47 0.23 * . . 0.38 0.71 Ile 27 A A . B . .. 0.34 −0.41 * . . 1.32 0.70 Pro 28 A . . . . T . 1.24 −0.41 . * F 2.361.65 Lys 29 . . . . T T . 0.90 −1.10 . . F 3.40 3.81 Arg 30 . . . . T T. 0.86 −1.50 . . F 3.06 7.29 Arg 31 . . . . T T . 1.24 −1.54 . . F 2.813.50 Lys 32 . . . B T . . 1.79 −1.49 . . F 2.16 2.52 Ser 33 . . . B . .C 2.00 −1.06 . * F 1.71 1.28 Val 34 . . . B . . C 1.07 −1.06 . . F 1.461.13 Thr 35 . . B B . . . 0.10 −0.37 . * F 0.90 0.40 Gly 36 . . B B . .. −0.82 0.27 . * F 0.21 0.22 Glu 37 . . B B . . . −1.76 0.57 * . F −0.180.24 Ile 38 . . B B . . . −1.77 0.61 . * . −0.42 0.12 Val 39 . . B B . .. −1.26 0.61 . . . −0.51 0.17 Leu 40 . . B B . . . −1.53 0.61 . . .−0.60 0.10 Ile 41 . . B B . . −1.53 1.11 . . . −0.60 0.14 Thr 42 . . B B. . . −1.57 0.86 . . . −0.60 0.19 Gly 43 . . B . . T . −1.02 0.71 * . .−0.20 0.31 Ala 44 . . . . . T C −1.06 0.46 . . . 0.00 0.44 Gly 45 . . .. . T C −0.59 0.46 * * . 0.00 0.21 His 46 . . . . . T C 0.41 0.40 * * .0.00 0.21 Gly 47 . . . . . . C −0.09 −0.03 * * . 0.70 0.42 Ile 48 . . B. . . . −0.06 0.16 * * . −0.10 0.35 Gly 49 . . B . . . . −0.06 0.21 * *. −0.10 0.37 Arg 50 . A B . . . . 0.04 0.21 * * . −0.30 0.37 Leu 51 A B. . . . 0.08 0.54 * * . −0.60 0.84 Thr 52 . A B . . . −0.28 −0.14 * . .0.45 1.46 Ala 53 A A . . . . . 0.02 0.21 * * . −0.30 0.65 Tyr 54 A A . .. . . 0.41 0.71 * * . −0.60 0.79 Glu 55 A A . . . . . −0.51 0.03 * * .−0.15 1.10 Phe 56 A A . . . . . 0.34 0.23 . * . −0.30 0.90 Ala 57 A A .. . . . 0.36 −0.27 * * . 0.45 1.15 Lys 58 A A . . . . . 0.99 −0.64 * * F0.75 0.89 Leu 59 A A . . . . . 0.42 −0.64 * * F 0.90 2.05 Lys 60 A A . .. . . −0.43 −0.74 * * F 0.90 1.67 Ser 61 A A . . . . . −0.54 −0.60 * * F0.75 0.62 Lys 62 A A . . . . . −0.24 0.09 . * F −0.15 0.62 Leu 63 A A .. . . . −0.29 0.31 . * F −0.15 0.33 Val 64 A A . . . . . −0.37 0.31 . *. −0.30 0.41 Leu 65 A A . . . . . −0.41 0.61 * . . −0.60 0.14 Trp 66 A A. . . . . −0.07 1.01 * . . −0.60 0.28 Asp 67 A A . . . . . −0.14 0.33 *. . −0.30 0.75 Ile 68 A A . . . . . 0.32 0.19 * . . 0.12 1.23 Asn 69 A .. . . T . 0.37 −0.07 * . . 1.39 1.16 Lys 70 . . . . . T C 1.18 −0.30 * .F 1.86 0.57 His 71 . . . . . T C 1.47 −0.30 * . . 2.13 1.42 Gly 72 . . .. . T C 1.16 −0.99 * . . 2.70 1.53 Leu 73 A A . . . . . 1.46 −0.90 . . F1.98 1.10 Glu 74 A A . . . . . 0.87 −0.40 . . F 1.26 0.82 Glu 75 A A . .. . . 0.87 −0.40 . . F 0.99 0.84 Thr 76 A A . . . . . 0.23 −0.83 * * F1.17 2.03 Ala 77 A A . . . . . 0.62 −0.94 * F 0.75 0.63 Ala 78 A A . . .. . 1.09 −0.94 . . . 0.60 0.72 Lys 79 A A . . . . . 0.28 −0.51 . * F0.75 0.50 Cys 80 A . . . . T . −0.07 −0.31 . * F 0.85 0.41 Lys 81 A . .. . T . −0.34 −0.39 . . F 0.85 0.40 Gly 82 A . . . . T . 0.29 −0.39 . *F 0.85 0.20 Leu 83 A . . . . T . 0.02 −0.39 * F 0.85 0.75 Gly 84 A . . .. . . −0.06 −0.31 . * . 0.50 0.28 Ala 85 A . . . . . . 0.30 0.19 * . .−0.10 0.38 Lys 86 A . . B . . . −0.44 0.24 * * . −0.30 0.67 Val 87 . . BB . . . −0.96 0.34 . * . −0.30 0.58 His 88 . . B B . . . −1.00 0.56 * *. −0.60 0.43 Thr 89 . . B B . . . −0.66 0.70 * . . −0.60 0.16 Phe 90 . .B B . . . −0.73 0.70 * * . −0.60 0.36 Val 91 . . B B . . . −1.08 0.63 *. . −0.26 0.14 Val 92 . . B B . . . −0.22 0.51 * * . 0.08 0.13 Asp 93 .. B B . . . −0.08 0.43 * * . 0.42 0.24 Cys 94 . . . . T T . 0.23−0.36 * * . 2.46 0.64 Ser 95 . . . . T T . 0.93 −1.00 . * F 3.40 1.50Asn 96 A . . . . T . 0.90 −1.64 . * F 2.66 1.50 Arg 97 A . . . . T .1.51 −0.96 * F 2.32 1.96 Glu 98 A . . . . . . 1.21 −0.77 . * F 1.78 2.29Asp 99 A . . . . . . 1.58 −0.77 . * F 1.44 1.91 Ile 100 A . . . . . .1.29 −0.79 . * . 0.95 1.31 Tyr 101 A . . . . T . 1.33 −0.29 * * . 0.700.76 Ser 102 A . . . . T . 1.27 −0.29 * * F 0.85 0.91 Ser 103 A . . . .T . 0.41 −0.29 * . F 1.00 2.61 Ala 104 A . . . . T . 0.46 −0.33 * * F1.00 1.23 Lys 105 A A . . . . . 0.76 −1.09 * * F 0.90 1.84 Lys 106 A A .. . . . 1.00 −0.97 * * F 0.90 1.39 Val 107 A A . . . . . 0.41 −1.36 * *F 0.90 2.38 Lys 108 A A . . . . . 0.37 −1.17 * * F 0.75 0.83 Ala 109 A A. . . . . 0.96 −0.74 * * F 0.75 0.41 Glu 110 A A . . . . . 0.06−0.74 * * F 0.75 0.93 Ile 111 A . . B . . . −0.29 −0.74 . * F 0.75 0.34Gly 112 A . . B . . . −0.32 −0.36 . * F 0.45 0.46 Asp 113 A . . B . . .−1.18 −0.17 . * F 0.45 0.19 Val 114 . . B B . . . −1.44 0.51 . * . −0.600.22 Ser 115 . . B B . . . −1.44 0.47 * . . −0.60 0.16 Ile 116 . . B B .. . −0.56 0.44 * . . −0.60 0.16 Leu 117 . . B B . . . −0.80 0.84 . * .−0.60 0.34 Val 118 . . B B . . . −1.14 0.70 . * . −0.60 0.26 Asn 119 . .B B . . . −1.14 0.74 . . −0.60 0.36 Asn 120 . . B . . T . −1.70 0.70 . .. −0.20 0.33 Ala 121 . . B . . T . −1.06 0.66 . . . −0.20 0.33 Gly 122 .. B . . T . −0.56 0.77 . . . −0.20 0.32 Val 123 . B . . T . 0.00 0.86 .. . −0.20 0.29 Val 124 . . B . . . . 0.00 0.84 . . . −0.40 0.38 Tyr 125. . B . . . . −0.81 0.34 . . . −0.10 0.64 Thr 126 . . B . . T . −0.920.60 . . F −0.05 0.71 Ser 127 . . B . . T . −1.17 0.74 . . F −0.05 0.83Asp 128 . . B . . T . −0.62 0.60 . . F −0.05 0.53 Leu 129 . . B . . T .0.23 0.33 . . F 0.25 0.53 Phe 130 . . B . . . . 0.48 0.24 . . . −0.100.69 Ala 131 A . . . . . . 0.58 −0.14 . . F 0.65 0.69 Thr 132 A . . . .T . 0.88 0.29 . * F 0.40 1.29 Gln 133 A . . . . T . −0.01 0.00 . * F0.40 2.59 Asp 134 . . . . . T C 0.80 −0.10 * . F 1.20 1.80 Pro 135 A . .. . T . 1.54 −0.60 * . F 1.30 2.16 Gln 136 A A . . . . . 1.82 −1.09 * *F 0.90 2.49 Ile 137 A A . . . . . 1.43 −1.00 * * F 0.90 2.15 Glu 138 A A. . . . . 1.43 −0.21 * * F 0.60 1.20 Lys 139 A A . . . . . 0.58−0.64 * * F 0.90 1.20 Thr 140 A . . B . . . 0.79 −0.40 * * F 0.60 1.28Phe 141 A . . B . . . −0.07 −0.69 * * . 0.75 1.18 Glu 142 A . . B . . .0.01 −0.04 . * . 0.30 0.44 Val 143 A . . B . . . −0.58 0.64 . * . −0.600.25 Asn 144 A . . B . . . −0.66 0.66 . * . −0.60 0.29 Val 145 A . . B .. . −1.04 0.37 . * . −0.30 0.23 Leu 146 A . . B . . . −0.63 1.16 . * .−0.60 0.27 Ala 147 A . . B . . . −0.94 1.43 * * . −0.60 0.18 His 148 A .. B . . . −0.40 1.51 . * . −0.60 0.34 Phe 149 A . . B . . . −0.36 1.36 .. . −0.60 0.60 Trp 150 A . . B . . . −0.09 0.67 . . . −0.45 1.18 Thr 151A . . B . . . 0.02 0.67 . . F −0.45 0.88 Thr 152 A . . B . . −0.20 0.96. . F −0.45 0.88 Lys 153 A . . B . . . −0.38 0.86 . . F −0.45 0.69 Ala154 A A . . . . . −0.27 0.37 . . . −0.30 0.74 Phe 155 A A . . . . .−0.58 0.39 . . . −0.30 0.52 Leu 156 A A . . . . . −0.58 0.51 * . . −0.600.26 Pro 157 A A . . . . . −0.22 1.00 * . . −0.60 0.37 Ala 158 A A . . .. . −0.27 0.50 * . . −0.60 0.84 Met 159 A A . . . . . 0.32 0.11 . . .0.09 1.64 Thr 160 A A . . . . . 0.99 −0.17 . . F 1.08 1.71 Lys 161 A A .. . . . 1.46 −0.10 . . F 1.32 2.30 Asn 162 A . . . . T . 1.63 −0.17 . .F 1.96 2.30 Asn 163 . . . . . T C 1.33 −0.29 . . F 2.40 2.17 His 164 . .. . . T C 1.08 −0.09 . . . 1.86 0.76 Gly 165 . . . . . T C 1.08 0.56 . .. 0.72 0.35 His 166 . . B B . . . 0.18 0.64 . . . −0.12 0.32 Ile 167 . BB . . . −0.41 0.89 . . . −0.36 0.17 Val 168 . . B B . . . −0.71 0.89 . .. −0.60 0.18 Thr 169 . . B B . . . −1.27 0.84 . . . −0.60 0.17 Val 170 .. B B . . . −1.51 0.84 . . . −0.60 0.25 Ala 171 A . . B . . . −1.820.66 * . . −0.60 0.34 Ser 172 A . . B . . . −0.97 0.44 * . . −0.60 0.23Ala 173 A . . . . . . −0.97 0.46 . . . −0.40 0.43 Ala 174 A . . B . . .−0.96 0.46 . * . −0.60 0.31 Gly 175 A . . B . . . −0.96 0.34 . * . −0.300.31 His 176 . . B B . . . −0.58 0.60 . * . −0.60 0.23 Val 177 . . B B .. . −0.98 0.53 . * . −0.60 0.35 Ser 178 . . B B . . −1.20 0.81 . * .−0.60 0.31 Val 179 . . B B . . −1.42 1.07 . * . −0.60 0.19 Pro 180 . B B. . . −1.67 1.26 . * . −0.60 0.21 Phe 181 . . B B . . . −1.88 1.11 . * .−0.60 0.16 Leu 182 . . B B . . . −1.69 1.49 . * . −0.60 0.33 Leu 183 A .. B . . . −1.69 1.41 . . . −0.60 0.11 Ala 184 A . B . . . −1.13 1.37 . .. −0.60 0.18 Tyr 185 A . . B . . . −0.88 0.97 . * . −0.60 0.29 Cys 186 A. . . . T . −0.88 0.29 . . . 0.10 0.70 Ser 187 A . . . . T . −0.66 0.39. * . 0.10 0.60 Ser 188 A . . . . T . −0.43 0.39 . . F 0.25 0.39 Lys 189A . . . . T . −0.70 0.13 . . F 0.25 0.73 Phe 190 A A . . . . −0.80 0.20. . . −0.30 0.40 Ala 191 A A . . . . . −0.83 0.24 . . . −0.30 0.30 Ala192 A A . . . . . −0.57 0.64 . . . −0.60 0.13 Val 193 A A . . . . .−0.22 1.14 * . . −0.60 0.20 Gly 194 A A . . . . . −0.58 0.36 * . . −0.300.40 Phe 195 A A . . . . . −0.69 0.34 . . . −0.30 0.57 His 196 A A . . .. . −0.41 0.53 * . . −0.60 0.64 Lys 197 A A . . . . . 0.18 0.37 * . F−0.15 0.93 Thr 198 A A . . . . . 1.03 −0.06 * . F 0.60 1.79 Leu 199 A A. . . . . 0.57 −0.84 * . F 0.90 2.28 Thr 200 A A . . . . . 0.68 −0.66 *. F 0.75 0.94 Asp 201 A A . . . . . 0.12 −0.16 * . F 0.45 0.66 Glu 202 AA . . . . −0.73 −0.14 * . . 0.30 0.81 Leu 203 A A . . . . . −0.42 −0.14. . . 0.30 0.46 Ala 204 A A . . . . . −0.50 −0.23 . . . 0.30 0.48 Ala205 A A . . . . . −0.50 0.46 . * . −0.60 0.19 Leu 206 A A . . . . .−0.84 0.94 . . . −0.60 0.34 Gln 207 A A . . . . . −1.70 0.69 . . . −0.600.33 Ile 208 A A . . . . . −0.84 0.83 . . . −0.60 0.24 Thr 209 . A B . .. −0.57 0.33 . . F −0.15 0.59 Gly 210 . A . . T . . −0.29 0.13 . . F0.25 0.49 Val 211 . . B B . . . −0.14 0.21 . . F 0.00 1.01 Lys 212 . . BB . . . −0.96 0.10 . . F −0.15 0.38 Thr 213 . . B B . . . −0.73 0.30 . .F −0.15 0.31 Thr 214 . . B B . . . −0.63 0.44 . . . −0.60 0.23 Cys 215 .. B B . . . −0.29 0.23 . . . −0.30 0.17 Leu 216 . . B B . . . −0.13 0.63. . . −0.60 0.19 Cys 217 . B . . T . −1.03 0.93 * . . −0.20 0.12 Pro 218. . B . . T . −0.72 1.09 * . −0.20 0.16 Asn 219 . . . . T T . −0.720.91 * . . 0.20 0.32 Phe 220 . . B . . T . −0.40 0.71 * . . −0.20 0.85Val 221 . . B . . . . −0.29 0.57 . . . −0.40 0.54 Asn 222 . . B . . T .−0.51 0.93 * . . −0.20 0.29 Thr 223 . . B . . T . −0.26 1.21 . . . −0.200.24 Gly 224 . . B . . T . −0.26 0.43 . . . −0.20 0.64 Phe 225 . . B . .T . 0.23 0.19 . . . 0.10 0.64 Ile 226 . . B . . . . 0.79 0.21 * . .−0.10 0.68 Lys 227 . . B . . . . 0.48 0.11 . . F 0.05 0.93 Asn 228 . . .. T C 0.49 0.17 * F 0.60 1.55 Pro 229 . . . . T T . 0.02 −0.23 * . F1.40 2.95 Ser 230 . . . . T T . 0.38 −0.23 * . F 1.40 1.22 Thr 231 . . .. T T . 1.06 0.20 * . F 0.65 0.75 Ser 232 . . . . . . C 0.70 0.23 * . F0.25 0.75 Leu 233 . . . . . . C −0.11 0.29 * * F 0.55 0.81 Gly 234 . . .. T C 0.10 0.59 . * F 0.75 0.46 Pro 235 . . . . . T C 0.19 0.10 . * F1.35 0.60 Thr 236 . . . . . T C 0.50 0.14 . . F 1.80 1.12 Leu 237 . . .. . T C 0.80 −0.54 . . F 3.00 1.96 Glu 238 . A B . . . . 0.76 −0.97 . .F 2.10 2.19 Pro 239 A A . . . . . 0.24 −0.76 . . F 1.80 1.13 Glu 240 A A. . . . . 0.46 −0.60 * . F 1.50 1.01 Glu 241 A A . . . . . 0.88−0.89 * * F 1.05 0.94 Val 242 A A . . . . 0.88 −0.89 * . . 0.75 1.19 Val243 A A . . . . . 0.28 −0.63 * . . 0.60 0.57 Asn 244 A A . . . . . 0.46−0.01 * . . 0.30 0.32 Arg 245 A A . . . . . 0.11 0.49 * . . −0.60 0.60Leu 246 A A . . . . . −0.78 0.27 * . . −0.30 0.79 Met 247 A A . . . . .−0.73 0.31 * . . −0.30 0.35 His 248 A A . . . . . −0.19 0.60 * . . −0.600.15 Gly 249 A A . . . . . −0.19 1.09 * . . −0.60 0.25 Ile 250 A A . . .. . −0.30 0.40 * . . −0.60 0.45 Leu 251 A A . . . . . 0.56 0.19 * . .−0.30 0.57 Thr 252 A A . . . . . 0.56 −0.31 * . F 0.60 1.15 Glu 253 A A. . . . . −0.30 −0.13 * . F 0.60 1.62 Gln 254 A A . . . . . −0.66 −0.13. . F 0.60 1.38 Lys 255 A A . . . . . −0.66 −0.03 . . F 0.45 0.83 Met256 . A B . . . . −0.06 0.17 . . . −0.30 0.33 Ile 257 . A B . . . .−0.04 0.60 . . −0.60 0.30 Phe 258 . A B . . . . −0.34 0.59 . . . −0.600.20 Ile 259 . . B . . T . −1.23 0.97 * . . −0.20 0.27 Pro 260 . . B . .T . −1.87 1.04 . . F −0.05 0.27 Ser 261 . . . . . T C −1.97 0.86 . . F0.15 0.32 Ser 262 . . . . . T C −1.89 0.86 . . F 0.15 0.39 Ile 263 A . .B . . . −1.50 0.86 . . . −0.60 0.21 Ala 264 A . . B . . . −0.92 0.91 * .. −0.60 0.22 Phe 265 A . . B . . . −1.52 1.01 . . . −0.60 0.24 Leu 266 A. . B . . . −1.22 1.31 * . . −0.60 0.28 Thr 267 A . . B . . . −0.810.63 * * . −0.60 0.49 Thr 268 A . . B . . . −0.81 0.13 * . F 0.00 1.10Leu 269 A . . B . . . −1.03 0.03 * . F −0.15 0.94 Glu 270 A . . B . . .−0.54 0.03 * . F −0.15 0.53 Arg 271 A . . B . . . 0.27 −0.03 * . . 0.300.57 Ile 272 A . B . . . 0.69 −0.51 * . . 0.75 1.20 Leu 273 A . . B . .. 0.30 −1.20 * . F 0.90 1.36 Pro 274 A . . B . . . 0.30 −0.41 * . F 0.450.60 Glu 275 A A . . . . . −0.29 0.27 * . F −0.15 0.71 Arg 276 A A . B .. . −1.26 0.09 * . . −0.30 0.87 Phe 277 A A . B . . . −1.18 0.04 * * .−0.30 0.42 Leu 278 A A . B . . . −0.32 0.30 * * . −0.30 0.20 Ala 279 A A. B . . . 0.00 0.30 * . . −0.30 0.20 Val 280 A A . B . . . 0.04 0.30 * *. −0.30 0.46 Leu 281 A A . B . . . −0.96 −0.49 * * . 0.45 1.11 Lys 282 AA . B . . . −0.56 −0.49 * * F 0.45 0.77 Arg 283 A A . B . . . −0.60−0.60 . * F 0.90 1.39 Lys 284 A A . B . . . 0.03 −0.60 . * F 0.90 1.25Ile 285 A A . B . . . 0.19 −1.29 * * F 0.90 1.25 Ser 286 . A B B . . .1.00 −0.50 * * . 0.30 0.55 Val 287 . A B B . . . 0.37 −0.50 * * . 0.300.46 Lys 288 . A B B . . . −0.60 0.00 * * . −0.30 0.67 Phe 289 . A B B .. . −1.53 −0.04 * * . 0.30 0.37 Asp 290 A A . B . . . −0.99 0.26 . * .−0.30 0.35 Ala 291 A A . B . . . −0.93 0.04 . * . −0.30 0.17 Val 292 A A. B . . . −0.03 0.80 . * . −0.60 0.31 Ile 293 A . . B . . . −0.680.01 * * . −0.30 0.37 Gly 294 A . . . . . . 0.07 0.63 . * . −0.40 0.37Tyr 295 A A . . . . . −0.52 0.13 . * . −0.30 0.99 Lys 296 A A . . . . .0.07 −0.01 . * . 0.45 1.42 Met 297 A A . . . . . 0.53 −0.30 . * F 0.602.49 Lys 298 A A . . . . . 1.03 −0.30 . * . 0.45 2.03 Ala 299 A A . . .. . 0.99 −0.63 . * . 0.75 1.30 Gln 300 A A . . . . . 0.84 −0.20 . * .0.45 1.68

1. An isolated protein comprising amino acid residues 19 to 300 of SEQID NO:98.
 2. The isolated protein of claim 1 which comprises amino acidresidues 2 to 300 of SEQ 1D NO:98.
 3. The isolated protein of claim 1which comprises amino acid residues 1 to 300 of SEQ ID NO:98.
 4. Theisolated protein of claim 3 which further comprises a heterologouspolypeptide sequence.
 5. The isolated protein of claim 4 wherein theheterologous polypeptide is the Fc domain of immunoglobulin.
 6. Acomposition comprising the isolated protein of claim 3 and a carrier. 7.An isolated protein produced by the method comprising: (a) expressingthe protein of claim 3 by a cell; and (b) recovering said protein. 8.The isolated protein of claim 3 wherein said isolated protein isglycosylated.
 9. An isolated protein comprising the amino acid sequenceof the secreted portion of the polypeptide encoded by the HAGDG59 cDNAcontained in American Type Culture Collection (“ATCC”) Deposit No.209277.
 10. The isolated protein of claim 9 which comprises the aminoacid sequence of the complete polypeptide encoded by the HAGDG59 cDNAcontained in ATCC Deposit No. 209277, excepting the N-terminalmethionine.
 11. The isolated protein of claim 9 which comprises theamino acid sequence of the complete polypeptide encoded by the HAGDG59cDNA contained in ATCC Deposit No.
 209277. 12. The isolated protein ofclaim 11 which further comprises a heterologous polypeptide sequence.13. The isolated protein of claim 12 wherein the heterologouspolypeptide is the Fc domain of immunoglobulin.
 14. A compositioncomprising the isolated protein of claim 11 and a carrier.
 15. Anisolated protein produced by the method comprising: (a) expressing theprotein of claim 11 by a cell; and (b) recovering said protein.
 16. Theisolated protein of claim 11 wherein said isolated protein isglycosylated.
 17. An isolated protein comprising a polypeptide which isat least 90% identical to amino acid residues 1 to 300 of SEQ ID NO:98,wherein said protein modulates the T-cell immune response during woundrepair.
 18. The isolated protein of claim 17, wherein said polypeptidesequence is at least 95% identical to amino acid residues 1 to 300 ofSEQ ID NO:98.
 19. The isolated protein of claim 17 which furthercomprises a heterologous polypeptide sequence.
 20. The isolated proteinof claim 19 wherein the heterologous polypeptide is the Fc domain ofimmunoglobulin.
 21. A composition comprising the isolated protein ofclaim 17 and a carrier.
 22. An isolated protein produced by the methodcomprising: (a) expressing the protein of claim 17 by a cell; and (b)recovering said protein.
 23. The isolated protein of claim 17 whereinsaid isolated protein is glycosylated.
 24. An isolated proteincomprising a polypeptide which is at least 90% identical to the completepolypeptide encoded by the HAGDG59 cDNA contained in ATCC Deposit No.209277, wherein said protein modulates the T-cell immune response duringwound repair.
 25. The isolated protein of claim 28, wherein saidpolypeptide sequence is at least 95% identical to the completepolypeptide encoded by the HAGDG59 cDNA contained in ATCC Deposit No.209277.
 26. The isolated protein of claim 24 which further comprises aheterologous polypeptide sequence.
 27. The isolated protein of claim 26wherein the heterologous polypeptide is the Fc domain of immunoglobulin.28. A composition comprising the isolated protein of claim 24 and acarrier.
 29. An isolated protein produced by the method comprising: (a)expressing the protein of claim 24 by a cell; and (b) recovering saidprotein.
 30. The isolated protein of claim 24 wherein said isolatedprotein is glycosylated.
 31. An isolated protein consisting of at least30 contiguous amino acid residues of amino acid residues 1 to 300 of SEQID NO:98, wherein said protein modulates the T-cell immune responseduring wound repair.
 32. The isolated protein of claim 31 which consistsof at least 50 contiguous amino acid residues of amino acid residues 1to 300 of SEQ ID NO:98.
 33. The isolated protein of claim 31 whichfurther comprises a heterologous polypeptide sequence.
 34. The isolatedprotein of claim 33 wherein the heterologous polypeptide is the Fcdomain of immunoglobulin.
 35. A composition comprising the isolatedprotein of claim 31 and a carrier.
 36. An isolated protein produced bythe method comprising: (a) expressing the protein of claim 31 by a cell;and (b) recovering said protein.
 37. The isolated protein of claim 31wherein said isolated protein is glycosylated.
 38. An isolated proteinconsisting of at least 30 contiguous amino acid residues of the completepolypeptide encoded by the HAGDG59 cDNA contained in ATCC Deposit No.209277, wherein said protein modulates the T-cell immune response duringwound repair.
 39. The isolated protein of claim 38 which consists of atleast 50 contiguous amino acid residues of the complete polypeptideencoded by the HAGDG59 cDNA contained in ATCC Deposit No.
 209277. 40.The isolated protein of claim 38 which further comprises a heterologouspolypeptide sequence.
 41. The isolated protein of claim 40 wherein theheterologous polypeptide is the Fc domain of immunoglobulin.
 42. Acomposition comprising the isolated protein of claim 38 and carrier. 43.An isolated protein produced by the method comprising: (a) expressingthe protein of claim 38 by a cell; and (b) recovering said protein. 44.The isolated protein of claim 38 wherein said isolated protein isglycosylated.